1. Mechanisms of Wound Healing and Fibrosis
The term “wound healing” refers to the process by which the body repairs trauma to any of its tissues, especially those caused by physical means and with interruption of continuity.
A wound-healing response often is described as having three distinct phases-injury, inflammation and repair. Generally speaking, the body responds to injury with an inflammatory response, which is crucial to maintaining the health and integrity of an organism. If however it goes awry, it can result in tissue destruction.
Phase I: Injury
Injury caused by factors including, but not limited to, autoimmune or allergic reactions, environmental particulates, infection or mechanical damage often results in the disruption of normal tissue architecture, initiating a healing response. Damaged epithelial and endothelial cells must be replaced to maintain barrier function and integrity and prevent blood loss, respectively. Acute damage to endothelial cells leads to the release of inflammatory mediators and initiation of an anti-fibrinolytic coagulation cascade, temporarily plugging the damaged vessel with a platelet and fibrin-rich clot. For example, lung homogenates, epithelial cells or bronchoalveolar lavage fluid from idiopathic pulmonary fibrosis (IPF) patients contain greater levels of the platelet-differentiating factor, X-box-binding protein-1, compared with chronic obstructive pulmonary disease (COPD) and control patients, suggesting that clot-forming responses are continuously activated. In addition, thrombin (a serine protease required to convert fibrinogen into fibrin) is also readily detected within the lung and intra-alveolar spaces of several pulmonary fibrotic conditions, further confirming the activation of the clotting pathway. Thrombin also can directly activate fibroblasts, increasing proliferation and promoting fibroblast differentiation into collagen-producing myofibroblasts. Damage to the airway epithelium, specifically alveolar pneumocytes, can evoke a similar anti-fibrinolytic cascade and lead to interstitial edema, areas of acute inflammation and separation of the epithelium from the basement membrane.
Platelet recruitment, degranulation and clot formation rapidly progress into a phase of vasoconstriction with increased permeability, allowing the extravasation (movement of white blood cells from the capillaries to the tissues surrounding them) and direct recruitment of leukocytes to the injured site. The basement membrane, which forms the extracellular matrix underlying the epithelium and endothelium of parenchymal tissue, precludes direct access to the damaged tissue. To disrupt this physical barrier, zinc-dependent endopeptidases, also called matrix metalloproteinases (MMPs), cleave one or more extracelluar matrix constituents allowing extravasation of cells into, and out of, damaged sites. Specifically, MMP-2 (gelatinase A, Type N collagenase) and MMP-9 (gelatinase B, Type IV collagenase) cleave type N collagens and gelatin, two important constituents of the basement membrane. Recent studies have found that MMP-2 and MMP-9 are upregulated, highlighting that tissue-destructive and regenerative processes are common in fibrotic conditions. The activities of MMPs are controlled by several mechanisms including transcriptional regulation, proenzyme regulation, and specific tissue inhibitors of MMPs. The balance between MMPs and the various inhibitory mechanisms can regulate inflammation and determine the net amount of collagen deposited during the healing response.
Previous studies using a model of allergic airway inflammation and remodeling with MMP-2−/−, MMP-9−/− and MMP-2−/− MMP-9−/− double knockout mice showed that MMP-2 and MMP-9 were required for successful egression and clearance of inflammatory cells out of the inflamed tissue and into the airspaces. In the absence of these MMPs, cells were trapped within the parenchyma of the lung and were not able to move into the airspaces, which resulted in fatal asphyxiation.
Phase II: Inflammation
Once access to the site of tissue damage has been achieved, chemokine gradients recruit inflammatory cells. Neutrophils, eosinophils, lymphocytes, and macrophages are observed at sites of acute injury with cell debris and areas of necrosis cleared by phagocytes.
The early recruitment of eosinophils, neutrophils, lymphocytes, and macrophages providing inflammatory cytokines and chemokines can contribute to local TGF-β and IL-13 accumulation. Following the initial insult and wave of inflammatory cells, a late-stage recruitment of inflammatory cells may assist in phagocytosis, in clearing cell debris, and in controlling excessive cellular proliferation, which together may contribute to normal healing. Late-stage inflammation may serve an anti-fibrotic role and may be required for successful resolution of wound-healing responses. For example, a late-phase inflammatory profile rich in phagocytic macrophages, assisting in fibroblast clearance, in addition to IL-10-secreting regulatory T cells, suppressing local chemokine production and TGF-β, may prevent excessive fibroblast activation.
The nature of the insult or causative agent often dictates the character of the ensuing inflammatory response. For example, exogenous stimuli like pathogen-associated molecular patterns (PAMPs) are recognized by pathogen recognition receptors, such as toll-like receptors and NOD-like receptors (cytoplasmic proteins that have a variety of functions in regulation of inflammatory and apoptotic responses), and influence the response of innate cells to invading pathogens. Endogenous danger signals also can influence local innate cells and orchestrate the inflammatory cascade.
The nature of the inflammatory response dramatically influences resident tissue cells and the ensuing inflammatory cells. Inflammatory cells themselves also propagate further inflammation through the secretion of chemokines, cytokines, and growth factors. Many cytokines are involved throughout a wound-healing and fibrotic response, with specific groups of genes activated in various conditions. For example, chronic allergic airway disease in asthmatics is associated commonly with elevated type-2 helper T cell (Th2) related cytokine profiles (including, but not limited to, interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-13 (IL-13), and interleukin-9 (IL-9)), whereas chronic obstructive pulmonary disease and fibrotic lung disease (such as idiopathic pulmonary fibrosis) patients more frequently present pro-inflammatory cytokine profiles (including, but not limited to, interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), and platelet-derived growth factors (PDGFs)). Each of these cytokines has been shown to exhibit significant pro-fibrotic activity, acting through the recruitment, activation and proliferation of fibroblasts, macrophages, and myofibroblasts.
Phase III: Tissue Repair and Contraction
The closing phase of wound healing consists of an orchestrated cellular re-organization guided by a fibrin (a fibrous protein that is polymerized to form a “mesh” that forms a clot over a wound site)-rich scaffold formation, wound contraction, closure and re-epithelialization. The vast majority of studies elucidating the processes involved in this phase of wound repair have come from dermal wound studies and in vitro systems.
Myofibroblast-derived collagens and smooth muscle actin (α-SMA) form the provisional extracellular matrix, with macrophage, platelet, and fibroblast-derived fibronectin forming a fibrin scaffold. Collectively, these structures are commonly referred to as granulation tissues. Primary fibroblasts or alveolar macrophages isolated from idiopathic pulmonary fibrosis patients produce significantly more fibronectin and α-SMA than control fibroblasts, indicative of a state of heightened fibroblast activation. It has been reported that IPF patients undergoing steroid treatment had similar elevated levels of macrophage-derived fibronectin as IPF patients without treatment. Thus, similar to steroid resistant IL-13-mediated myofibroblast differentiation, macrophage-derived fibronectin release also appears to be resistant to steroid treatment, providing another reason why steroid treatment may be ineffective. From animal models, fibronectin appears to be required for the development of pulmonary fibrosis, as mice with a specific deletion of an extra type III domain of fibronectin (EDA) developed significantly less fibrosis following bleomycin administration compared with their wild-type counterparts.
In addition to fibronectin, the provisional extracellular matrix consists of glycoproteins (such as PDGF), glycosaminoglycans (such as hyaluronic acid), proteoglycans and elastin. Growth factor and TGF-β-activated fibroblasts migrate along the extracellular matrix network and repair the wound. Within skin wounds, TGF-β also induces a contractile response, regulating the orientation of collagen fibers. Fibroblast to myofibroblast differentiation, as discussed above, also creates stress fibers and the neo-expression of α-SMA, both of which confer the high contractile activity within myofibroblasts. The attachment of myofibroblasts to the extracellular matrix at specialized sites called the “fibronexus” or “super mature focal adhesions” pull the wound together, reducing the size of the lesion during the contraction phase. The extent of extracellular matrix laid down and the quantity of activated myofibroblasts determines the amount of collagen deposition. To this end, the balance of matrix metalloproteinases (MMPs) to tissue inhibitor of metalloproteinases (TIMPs) and collagens to collagenases vary throughout the response, shifting from pro-synthesis and increased collagen deposition towards a controlled balance, with no net increase in collagen. For successful wound healing, this balance often occurs when fibroblasts undergo apoptosis, inflammation begins to subside, and granulation tissue recedes, leaving a collagen-rich lesion. The removal of inflammatory cells, and especially α-SMA-positive myofibroblasts, is essential to terminate collagen deposition. Interestingly, in idiopathic pulmonary fibrosis patients, the removal of fibroblasts can be delayed, with cells resistant to apoptotic signals, despite the observation of elevated levels of pro-apoptotic and FAS-signaling molecules. This relative resistance to apoptosis may potentially underlie this fibrotic disease. However, several studies also have observed increased rates of collagen-secreting fibroblast and epithelial cell apoptosis in idiopathic pulmonary fibrosis, suggesting that yet another balance requires monitoring of fibroblast apoptosis and fibroblast proliferation. From skin studies, re-epithelialization of the wound site re-establishes the barrier function and allows encapsulated cellular re-organization. Several in vitro and in vivo models, using human or rat epithelial cells grown over a collagen matrix, or tracheal wounds in vivo, have been used to identify significant stages of cell migration, proliferation, and cell spreading. Rapid and dynamic motility and proliferation, with epithelial restitution from the edges of the denuded area occur within hours of the initial wound. In addition, sliding sheets of epithelial cells can migrate over the injured area assisting wound coverage. Several factors have been shown to regulate re-epithelialization, including serum-derived transforming growth factor alpha (TGF-α), and matrix metalloproteinase-7 (MMP-7) (which itself is regulated by TIMP-1).
Collectively, the degree of inflammation, angiogenesis, and amount of extracellular matrix deposition all contribute to ultimate development of a fibrotic lesion. Thus, therapeutic intervention that interferes with fibroblast activation, proliferation, or apoptosis requires a thorough understanding and appreciation of all of the phases of wound repair. Although these three phases are often presented sequentially, during chronic or repeated injury these processes function in parallel, placing significant demands on regulatory mechanisms. (Wilson and Wynn, Mucosal Immunol., 2009, 3(2):103-121).
2. Fibrosis as a Pathology
Fibrosis represents the formation or development of excess fibrous connective tissue in an organ or tissue, which is formed as a consequence of the normal or abnormal/reactive wound healing response leading to a scar. Fibrosis is characterized by, for example, without limitation, an aberrant deposition of an extracellular matrix protein, an aberrant promotion of fibroblast proliferation, an aberrant induction of differentiation of a population of fibroblasts into a population of myofibroblasts, an aberrant promotion of attachment of myofibroblasts to an extracellular matrix, or a combination thereof.
Pro-Inflammatory Mediators
Accumulating evidence has suggested that polypeptide mediators known as cytokines, including various lymphokines, interleukins, and chemokines, are important stimuli to collagen deposition in fibrosis. Released by resident tissue cells and recruited inflammatory cells, cytokines are thought to stimulate fibroblast proliferation and increased synthesis of extracellular matrix proteins, including collagen. For example, an early feature in the pathogenesis of idiopathic pulmonary fibrosis is alveolar epithelial and/or capillary cell injury. This promotes recruitment into the lung of circulating immune cells, such as monocytes, neutrophils, lymphocytes and eosinophils. These effector cells, together with resident lung cells, such as macrophages, alveolar epithelial and endothelial cells, then release cytokines, which stimulate target cells, typically fibroblasts, to replicate and synthesize increased amounts of collagen. Breakdown of extracellular matrix protein also may be inhibited, thereby contributing to the fibrotic process. (Coker and Laurent, Eur Respir J, 1998; 11:1218-1221)
Numerous cytokines have been implicated in the pathogenesis of fibrosis, including, without limitation, transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), endothelin-1 (ET-1) and the interleukins, interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-17 (IL-17). Chemokine leukocyte chemoattractants, including the factor Regulated upon Activation in Normal T-cells, Expressed and Secreted (RANTES), are also thought to play an important role. Elevated levels of pro-inflammatory cytokines, such as Interleukin 8 (IL-8), as well as related downstream cell adhesion molecules (CAMs) such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinases such as matrix metalloproteinase-7 (MMP-7), and signaling molecules such as S100 calcium-binding protein A12 (S100A12, also known as calgranulin C), in the peripheral blood have been found to be associated with mortality, lung transplant-free survival, and disease progression in patients with idiopathic pulmonary fibrosis (Richards et al, Am J Respir Crit Care Med, 2012, 185: 67-76).
The TGF-β family of proteins has a potent stimulatory effect on extracellular matrix deposition, and in fact has been used in constructing induced animal models of fibrosis through gene transfer. In vitro studies show that TGF-β1, secreted as a latent precursor, promotes fibroblast procollagen gene expression and protein synthesis. The data suggest that the other mammalian isoforms, TGF-β2 and TGF-β3, also stimulate human lung fibroblast collagen synthesis and reduce breakdown in vitro. In animal models of pulmonary fibrosis, enhanced TGF-β1 gene expression is temporally and spatially related to increased collagen gene expression and protein deposition. TGF-β1 antibodies reduce collagen deposition in murine bleomycin-induced lung fibrosis, and human fibrotic lung tissue shows enhanced TGF-β1 gene and protein expression.
TNF-α can stimulate fibroblast replication and collagen synthesis in vitro, and pulmonary TNF-α gene expression rises after administration of bleomycin in mice. Soluble TNF-α receptors reduce lung fibrosis in murine models, and pulmonary overexpression of TNF-α in transgenic mice is characterized by lung fibrosis. In patients with IPF or asbestosis (a chronic inflammatory and fibrotic medical condition affecting the parenchymal tissue of the lungs caused by the inhalation and retention of asbestos fibers), bronchoalveolar lavage fluid-derived macrophages release increased amounts of TNF-α compared with controls.
Endothelin (ET-1) also fulfills the criteria for a profibrotic cytokine. This molecule promotes fibroblast proliferation and chemotaxis and stimulates procollagen production. It is present in the lungs of patients with pulmonary fibrosis, and a recent report suggests that the ET-1 receptor antagonist, bosentan, ameliorates lung fibrosis when administered to experimental animals.
Unchecked Myofibroblast Proliferation/Activation and Fibrotic Foci Formation
Differentiation of fibroblasts into myofibroblasts has long been believed to be an important event in many conditions, including wound repair and fibrosis. For example, it has been reported that myofibroblasts occur in areas of active fibrosis and are responsible for production and deposition of extracellular matrix (ECM) proteins in pulmonary fibrosis. (Liu, T. et al., Am J Respir Cell Mol Biol, 2007, 37:507-517).
One hypothesis for the causation of idiopathic pulmonary fibrosis suggests that a still-unidentified stimulus produces repeated episodes of acute lung injury. Wound healing at these sites of injury ultimately leads to fibrosis, with loss of lung function. Fibroblast foci, the hallmark lesions of idiopathic pulmonary fibrosis, feature vigorous replication of mesenchymal cells and exuberant deposition of fresh extracellular matrix. Such foci are typical of alveolar epithelial-cell injury, with endoluminal plasma exudation and collapse of the distal air space. Mediators normally associated with wound healing, such as transforming growth factor-β1 (TGF-β1) and connective-tissue growth factor, are expressed also at these sites. The driving force for this focal acute lung injury and wound repair is unknown. 3. Disease or Conditions in which Fibrosis Plays a Role
Fibrosis has been implicated in a number of heterogeneous diseases or conditions, including, but not limited to, interstitial lung disease, such as idiopathic pulmonary fibrosis, acute lung injury (ALI), radiation-induced fibrosis, transplant rejection, liver fibrosis, renal fibrosis and vascular fibrosis.
3.1. Idiopathic Pulmonary Fibrosis (IPF)
Idiopathic Pulmonary fibrosis (IPF, also known as cryptogenic fibrosing alveolitis, CFA, or Idiopathic Fibrosing Interstitial Pneumonia) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia of uncertain etiology that occurs primarily in older adults, is limited to the lungs, and is associated with the radiologic and histological pattern of usual interstitial pneumonia (UIP) (Raghu G. et al., Am J Respir Crit Care Med., 183(6):788-824, 2011; Thannickal, V. et al., Proc Am Thorac Soc., 3(4):350-356, 2006). It may be characterized by abnormal and excessive deposition of fibrotic tissue in the pulmonary interstitium. On high-resolution computed tomography (HRCT) images, UIP is characterized by the presence of reticular opacities often associated with traction bronchiectasis. As IPF progresses, honeycombing becomes more prominent (Neininger A. et al., J Biol Chem., 277(5):3065-8, 2002). Pulmonary function tests often reveal restrictive impairment and reduced diffusing capacity for carbon monoxide (Thomas, T. et al., J Neurochem., 105(5): 2039-52, 2008). Studies have reported significant increases in TNF-α and IL-6 release in patients with idiopathic pulmonary fibrosis (IPF) (Zhang, Y, et al. J. Immunol. 150(9):4188-4196, 1993), which has been attributed to the level of expression of IL-1β (Kolb, M., et al. J. Clin. Invest, 107(12):1529-1536, 2001). The onset of IPF symptoms, shortness of breath and cough, are usually insidious but gradually progress, with death occurring in 70% of patients within five years after diagnosis. This grim prognosis is similar to numbers of annual deaths attributable to breast cancer (Raghu G. et al., Am J Respir Crit Care Med., 183(6):788-824, 2011).
IPF afflicts nearly 130,000 patients in the United States, with approximately 50,000 new patients annually and nearly 40,000 deaths each year worldwide (Raghu G. et al., Am J Respir Crit Care Med., 183(6):788-824, 2011). While these data are notable, a recent study reported that IPF may be 5-10 times more prevalent than previously thought, perhaps due to increasing prevalence or enhanced diagnostic capabilities (Thannickal, V. et al., Proc Am Thorac Soc., 3(4):350-356, 2006). Lung transplantation is considered a definitive therapy for IPF, but the five year survival post lung transplantation is less than 50%. Accordingly, even lung transplantation cannot be considered a “cure” for IPF. In addition to the physical and emotional toll on the patient, IPF is extremely expensive to treat and care for with national healthcare costs to in the range of $2.8 billion dollars for every 100,000 patients annually.
In addition, previous studies have suggested that superimposed environmental insults may be important in the pathogenesis of idiopathic pulmonary fibrosis. In most reported case series, up to 75 percent of index patients with idiopathic pulmonary fibrosis are current or former smokers. In large epidemiologic studies, cigarette smoking has been strongly associated with idiopathic pulmonary fibrosis. In addition, many of the inflammatory features of idiopathic pulmonary fibrosis are more strongly linked to smoking status than to the underlying lung disease. Thus, cigarette smoking may be an independent risk factor for idiopathic pulmonary fibrosis. Latent viral infections, especially those of the herpes virus family, have also been reported to be associated with idiopathic pulmonary fibrosis.
Since there is no known effective treatment for IPF, including lung transplantation, there remains a critical need for the development of novel therapeutics. There are a variety of therapeutic approaches currently being investigated, including anti-fibrotic therapies that may slow or inhibit the body's ability to produce scar or fibrotic tissue and pulmonary vasodilators to increase the tissue area for gas exchange in the lung. Aside from lung transplantation, potential IPF treatments have included corticosteroids, azathioprine, cyclophosphamide, anticoagulants, and N-acetylcysteine (Raghu G. et al., Am J Respir Crit Care Med., 183(6):788-824, 2011). In addition, supportive therapies such as oxygen therapy and pulmonary rehabilitation are employed routinely. However, none of these have definitely impacted the long term survival of IPF patients, which further highlights the unmet medical need for treatment options in IPF. As an example, despite mixed clinical program results, InterMune's oral small-molecule Esbriet® (pirfenadone) received European and Japanese approvals for patients with IPF. Esbriet® thus became the first medication specifically indicated for the treatment of IPF; due to equivocal trial outcomes and drug side effects, the drug's utility is viewed with skepticism in the United States, and did not receive an FDA approval based on the data submitted at that time. Accordingly, a large phase 3 clinical trial is in progress to determine its efficacy to support a New Drug Application in the United States.
Histopathologically, IPF can be described as accumulation of activated myofibroblasts (or mesenchymal cells) in fibroblastic foci (Thannickal, V. et al., Proc Am Thorac Soc., 3(4):350-356, 2006). Impaired apoptosis of myofibroblasts may result in a persistent and dysregulated repair process that culminates in tissue fibrosis. Arguably, inflammation also plays a critical role in IPF, perhaps through cyclic acute stimulation of fibroblasts. These findings point to potential targets for therapeutic intervention.
3.1.1. Pathogenesis of Idiopathic Pulmonary Fibrosis (IPF)
While pathogenic mechanisms are incompletely understood, the currently accepted paradigm proposes that injury to the alveolar epithelium is followed by a burst of pro-inflammatory and fibroproliferative mediators that invoke responses associated with normal tissue repair. For unclear reasons, these repair processes never resolve and progressive fibrosis ensues. (Selman M, et al., Ann Intern Med, 134(2):136-151, 2001; Noble, P. and Homer R., Clin Chest Med, 25(4):749-58, 2004; Strieter, R., Chest, 128 (5 Suppl 1):526S-532S, 2005).
3.1.2. Bleomycin Mouse Model of Pulmonary Fibrosis
Although a number of animal models exist and can be useful (e.g., the TGF-β adenovirus transduction model or the radiation-induced fibrosis model), the bleomycin model is well-documented and the best characterized murine model in use today to demonstrate efficacy of a particular drug or protein kinase inhibitor in the post-inflammatory/pre-fibrotic/fibro-preventive stages (Vittal, R. et al., J Pharmacol Exp Ther., 321(1):35-44, 2007; Vittal, R. et al., Am J Pathol., 166(2):367-75, 2005; Hecker L. et al., Nat Med., 15(9):1077-81, 2009).
The antibiotic bleomycin was originally isolated from Streptomyces verticillatus (Umezawa, H. et al., Cancer 20: 891-895, 1967). This antibiotic was subsequently found to be effective against squamous cell carcinomas and skin tumors (Umezawa, H., Fed Proc, 33: 2296-2302, 1974); however, its usefulness as an anti-neoplastic agent was limited by dose-dependent pulmonary toxicity resulting in fibrosis (Muggia, F. et al., Cancer Treat Rev, 10: 221-243, 1983). The delivery of bleomycin via the intratracheal route (generally 1.25-4 U/kg, depending on the source) has the advantage that a single injection of the drug produces lung injury and resultant fibrosis in rodents (Phan, S. et al., Am Rev Respir Dis 121: 501-506, 1980; Snider, G. et al., Am Rev Respir Dis. 117: 289-297, 1978; Thrall, R. et al., Am J Pathol, 95: 117-130, 1979). Intratracheal delivery of the drug to rodents results in direct damage initially to alveolar epithelial cells. This event is followed by the development of neutrophilic and lymphocytic pan-alveolitis within the first week (Janick-Buckner, D. et al., Toxicol Appl Pharmacol., 100(3):465-73, 1989). Subsequently, alveolar inflammatory cells are cleared, fibroblast proliferation is noted, and extracellular matrix is synthesized (Schrier D. et al., Am Rev Respir Dis., 127(1):63-6, 1983). The development of fibrosis in this model can be seen biochemically and histologically by day 14 with maximal responses generally noted around days 21-28 (Izbicki G. et al., Int J Exp Pathol., 83(3):111-9, 2002; Phan, S. et al., Chest., 83(5 Suppl):44S-45S, 1983). Beyond 28 days, however, the response to bleomycin is more variable. Original reports suggest that bleomycin delivered intratracheally may induce fibrosis that progresses or persists for 60-90 days (Thrall R. et al., Am J Pathol., 95(1):117-30, 1979; Goldstein R., et al., Am Rev Respir Dis., 120(1):67-73, 1979; Starcher B. et al., Am Rev Respir Dis., 117(2):299-305, 1978); however, other reports demonstrate a self-limiting response that begins to resolve after this period (Thrall R. et al., Am J Pathol., 95(1):117-30, 1979; Phan, S. et al., Chest, 83(5 Suppl): 44S-45S, 1983; Lawson W. et al., Am J Pathol. 2005; 167(5):1267-1277). While the resolving nature of this model does not mimic human disease, this aspect of the model offers an opportunity for studying fibrotic resolution at these later time points.
3.2. Acute Lung Injury (ALI)
Acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), are syndromes of acute respiratory failure that result from acute pulmonary edema and inflammation. ALI/ARDS is a cause of acute respiratory failure that develops in patients of all ages from a variety of clinical disorders, including sepsis (pulmonary and nonpulmonary), pneumonia (bacterial, viral, and fungal), aspiration of gastric and oropharyngeal contents, major trauma, and several other clinical disorders, including severe acute pancreatitis, drug over dose, and blood products (Ware, L. and Matthay, M., N Engl J Med, 342:1334-1349, 2000). Most patients require assisted ventilation with positive pressure. The primary physiologic abnormalities are severe arterial hypoxemia as well as a marked increase in minute ventilation secondary to a sharp increase in pulmonary dead space fraction. Patients with ALI/ARDS develop protein-rich pulmonary edema resulting from exudation of fluid into the interstitial and airspace compartments of the lung secondary to increased permeability of the barrier. Additional pathologic changes indicate that the mechanisms involved in lung edema are complex and that edema is only one of the pathophysiologic events in ALI/ARDS. One physiologic consequence is a significant decrease in lung compliance that results in an increased work of breathing (Nuckton T. et al., N Engl J Med, 346:1281-1286, 2002), one of the reasons why assisted ventilation is required to support most patients.
It was suggested that mechanical ventilation (MV), a mainstay treatment for ALI, potentially contributes to and worsens permeability by exacting mechanical stress on various components of the respiratory system causing ventilator-associated lung injury (VALI) (Fan, E. et al., JAMA, 294:2889-2896, 2005; MacIntyre N., Chest, 128:561S-567, 2005). A recent trial demonstrated a significant improvement in survival in patients ventilated with low (LVT) compared to high tidal volumes (HVT) (The Acute Respiratory Distress Syndrome N. Ventilation with Lower Tidal Volumes as Compared with Traditional Tidal Volumes for Acute Lung Injury and the Acute Respiratory Distress Syndrome. N Engl J Med; 342:1301-1308, 2000). Other than ventilating at lower tidal volumes, which presumably imparts lower mechanical stress, there is little mechanistic understanding of the pathophysiology and no directed therapies for VALI.
It was suggested that the high tidal volumes (HVT) mechanical ventilation (MV) results in phosphorylation of p38 MAP kinase, activation of MK2, and phosphorylation of HSPB1, a process that causes actin to disassociate from HSPB1 and polymerize to form stress fibers, which ultimately leads to paracellular gaps and increased vascular permeability. Furthermore, it was shown that inhibiting p38 MAP kinase or its downstream effector MK2 prevents the phosphorylation of HSPB1 and protects from vascular permeability by abrogating actin stress fiber formation and cytoskeletal rearrangement, suggesting that targeted inhibition of MK2 could be a potential therapeutic strategy for the treatment of acute lung injury (Damarla, M. et al., PLoS ONE, 4(2): E4600, 2009).
Moreover, studies have suggested that pulmonary fibrosis can also result from ALI. ALI may completely resolve or proceed to fibrosing alveolitis accompanied by persistent low oxygen in the blood (hypoxemia) and a reduced ability of the lung to expand with every breath (reduced pulmonary compliance). It was suggested that while the etiology of injury-induced lung fibrosis is different from idiopathic pulmonary fibrosis, both diseases share a common pathological mechanism, i.e., infiltration of fibroblasts into the airspaces of lung (Tager et al., Nat. Med. 14: 45-54, 2008; Ley, K. and Zarbock, A., Nat. Med. 14: 20-21; 2008).
3.3. Radiation-Induced Fibrosis
Fibrosis is a common sequela of both cancer treatment by radiotherapy and accidental irradiation. Fibrotic lesions following radiotherapy have been described in many tissues, including skin (Bentzen, S. et al., Radiother. Oncol. 15: 261-214, 1989; Brocheriou, C., et al., Br. J. Radiol. Suppl. 19: 101-108, 1986), lung (Lopez Cardozo, B. et al., Int. J. Radiat. Oncol. Biol. Phys., 11: 907-914, 1985), heart (Fajardo, L. and Stewart, J., Lab. Invest., 29: 244-257, 1973), and liver (Ingold, J. et al., Am. J. Roentgenol., 93: 200-208, 1965).
In the lung (late responding tissue), two radiation toxicity syndromes, radiation pneumonitis and pulmonary fibrosis, may occur. Pneumonitis is manifested 2-3 months after radiotherapy is completed. Pathologically, pneumonitis is characterized by interstitial edema, the presence of interstitial and alveolar inflammatory cells, and an increase in the number of type II pneumocytes (Gross, N. et al., Radiat. Res., III: 143-50, 1981; Guerry-Force, M. et al., Radiat. Res. 114: 138-53, 1988). In pneumonitis, the primary damage to the tissue is most likely caused by depletion of parenchymal cells (Hendry, J., Radiat. Oncol. Vol. 4, 2: 123-132, 1994; Rosiello, R. et al., Am. Rev. Respir. Dis., 148: 1671-1676, 1993; Travis, E. and Terry, N., Front. Radiat. Ther. Oncol., 23: 41-59, 1989).
The fibrotic reaction is typified by increased interstitial collagen deposition, thickening of vascular walls and vascular occlusions (Vergava, J. et al., Int. J. Radiat. Oncol. Biol. Phys. 2: 723-732, 1987). Histological examinations of fibrotic lesions have revealed that fibrotic tissue contains infiltrating inflammatory cells, fibroblasts, and larger amounts of various extracellular matrix components. In fibrotic tissues, an enhanced synthesis and deposition of the interstitial collagens, fibronectin, and proteoglycans have been described (Maasiha, P. et al., Int. J. Radiat. Oncol. Biol. Phys. 20: 973-980, 1991), and this has been interpreted as the result of the radiation-induced modulation of the fibroblast cell system (Remy, J. et al., Radiat. Res. 125: 14-19, 1991).
Radiation-induced fibrosis, especially of the lung, was suggested to be due to an interplay of cellular and molecular events between several cell systems engaged in a fibrotic reaction. Irradiation alone is able to induce a premature terminal differentiation process of the fibroblast/fibrocyte cell system resulting in the enhanced accumulation of postmitotic fibrocytes, which are characterized by a several-fold increase in the synthesis of interstitial collagens. Concomitantly, irradiation of accompanying parenchymal cell types, such as alveolar macrophages and alveolar type II pneumocytes, induces the immediate synthesis of specific cytokines, like TGF-β1, which then alter the interaction of the parenchymal cells with the fibroblast cell system. TGF-β1, as one of the major cytokines responsible for the fibrotic reaction, induces the fibroblast proliferation via an expansion of the progenitor fibroblast cell types as well as a premature terminal differentiation of progenitor fibroblasts into post-mitotic fibrocytes. This leads to an accumulation of post-mitotic fibrocytes due to a disturbance of the well-balanced cell type ratio of progenitor fibroblasts and post-mitotic fibrocytes. It was proposed that the pathophysiological tissue response following irradiation is caused by an altered cytokine- and growth factor-mediated interaction of multicellular cell systems resulting in the disturbance of the well-balanced cell type ratio of the interstitial fibroblast/fibrocyte cell system. (Rodemann, H. and Bamberg, M., Radiotherapy and Oncology, 35, 83-90, 1995).
3.4. Transplant Rejection
Transplantation is the act of transferring cells, tissues, or organs from one site to another. The malfunction of an organ system can be corrected with transplantation of an organ (e.g., kidney, liver, heart, lung, or pancreas) from a donor. However, the immune system remains the most formidable barrier to transplantation as a routine medical treatment, and rejection of such organ often corresponds to a fibrotic phenotype in the grafted organ. The immune system has developed elaborate and effective mechanisms to combat foreign agents. These mechanisms are also involved in the rejection of transplanted organs, which are recognized as foreign by the host's immune system.
The degree of immune response to a graft depends partly on the degree of genetic disparity between the grafted organ and the host. Xenografts, which are grafts between members of different species, have the most disparity and elicit the maximal immune response, undergoing rapid rejection. Autografts, which are grafts from one part of the body to another (e.g., skin grafts), are not foreign tissue and, therefore, do not elicit rejection. Isografts, which are grafts between genetically identical individuals (e.g., monozygotic twins), also undergo no rejection.
Allografts are grafts between members of the same species that differ genetically. This is the most common form of transplantation. The degree to which allografts undergo rejection depends partly on the degree of similarity or histocompatibility between the donor and the host.
The degree and type of response also vary with the type of the transplant. Some sites, such as the eye and the brain, are immunologically privileged (i.e., they have minimal or no immune system cells and can tolerate even mismatched grafts). Skin grafts are not initially vascularized and so do not manifest rejection until the blood supply develops. The lungs, heart, kidneys, and liver are highly vascular organs and often lead to a vigorous cell mediated response in the host, requiring immunosuppressive therapies.
Constrictive bronchiolitis (CB), also termed in lung transplant patients obliterative bronchiolitis, is inflammation and fibrosis occurring predominantly in the walls and contiguous tissues of membranous and respiratory bronchioles with resultant narrowing of their lumens. CB is found in a variety of settings, most often as a complication of lung and heart-lung transplantation (affecting 34% to 39% of patients, usually in the first 2 years after transplantation) and bone marrow transplantation, but also in rheumatoid arthritis, after inhalation of toxic agents such as nitrogen dioxide, after ingestion of certain drugs such as penicillamine and ingestion of the East Asian vegetable Sauropus androgynous, and as a rare complication of adenovirus, influenza type A, measles, and Mycoplasma pneumoniae infections in children. In lung transplants, CB is the single most important factor leading to death thereafter. In one study, the overall mortality rate was 25%. However, at the same time, 87% of patients who were asymptomatic and diagnosed solely by transbronchial biopsy had resolution or stabilization of disease. Decreases in FEV1 from baseline can be used to clinically support CB in transplant patients; the term bronchiolitis obliterans syndrome is used to denote this clinical dysfunction, and a grading system has been established for it that is now widely used in the literature. Significant risk factors for the development of CB in lung transplants include alloantigen-dependent and -independent mechanisms. In the former group are late acute rejection and HLA mismatches at the A loci; in the latter are ischemia/reperfusion injuries to airways that result from the transplantation surgery and cytomegalovirus infection (Schlesinger C. et al, Curr Opin Pulm. Med., 4(5): 288-93, 1998).
Mechanisms of Rejection
The immune response to a transplanted organ consists of both cellular (lymphocyte mediated) and humoral (antibody mediated) mechanisms. Although other cell types are also involved, the T cells are central in the rejection of grafts. The rejection reaction consists of the sensitization stage and the effector stage.
Sensitization Stage
In this stage, the CD4 and CD8 T cells, via their T-cell receptors, recognize the alloantigens expressed on the cells of the foreign graft. Two signals are needed for recognition of an antigen; the first is provided by the interaction of the T cell receptor with the antigen presented by MHC molecules, the second by a co-stimulatory receptor/ligand interaction on the T cell/APC surface. Of the numerous co-stimulatory pathways, the interaction of CD28 on the T cell surface with its APC surface ligands, B7-1 or B7-2 (commonly known as CD80 or CD86, respectively), has been studied the most (Clarkson, M. and Sayegh, M., Transplantation; 80(5): 555-563, 2005). In addition, cytotoxic T lymphocyte-associated antigen-4 (CTLA4) also binds to these ligands and provides an inhibitory signal. Other co-stimulatory molecules include CD40 and its ligand CD40L (CD154). Typically, helices of the MHC molecules form the peptide-binding groove and are occupied by peptides derived from normal cellular proteins. Thymic or central tolerance mechanisms (clonal deletion) and peripheral tolerance mechanisms (e.g., anergy) ensure that these self-peptide MHC complexes are not recognized by the T cells, thereby preventing autoimmune responses.
Effector Stage
Alloantigen-dependent and independent factors contribute to the effector mechanisms. Initially, nonimmunologic “injury responses” (ischemia) induce a nonspecific inflammatory response. Because of this, antigen presentation to T cells is increased as the expression of adhesion molecules, class II MHC, chemokines, and cytokines is upregulated. It also promotes the shedding of intact, soluble MHC molecules that may activate the indirect allorecognition pathway. After activation, CD4-positive T cells initiate macrophage-mediated delayed type hypersensitivity (DTH) responses and provide help to B cells for antibody production.
Various T cells and T cell-derived cytokines such as IL-2 and IFN-γ are upregulated early after transplantation. Later, β-chemokines like RANTES (regulated upon activation, normal T cell expressed and secreted), IP-10, and MCP-1 are expressed, and this promotes intense macrophage infiltration of the allograft. IL-6, TNF-α, inducible nitric oxide synthase (iNOS) and growth factors, also play a role in this process. Growth factors, including TGF-β and endothelin, cause smooth muscle proliferation, intimal thickening, interstitial fibrosis, and, in the case of the kidney, glomerulosclerosis.
Endothelial cells activated by T cell-derived cytokines and macrophages express class II MHC, adhesion molecules, and co-stimulatory molecules. These can present antigen and thereby recruit more T cells, amplifying the rejection process. CD8-positive T cells mediate cell-mediated cytotoxicity reactions either by delivering a “lethal hit” or, alternatively, by inducing apoptosis.
In addition, emerging studies have suggested involvement of fibrotic processes in chronic transplant rejection of an organ transplant. For example, it was shown that chronic lung allograft rejection is mediated by a relative deficiency of allograft endothelial cell-derived HIF-1α, leading to fibrotic remodeling of the transplanted organ (Wilkes, D., J Clin Invest., 121(6): 2155-2157, 2011; Jiang, X. et al., J Clin Invest., 121(6): 2336-2349, 2011).
3.5. Chronic Obstructive Pulmonary Disease (COPD)
Chronic obstructive pulmonary disease (COPD) is a collective description for lung diseases represented by chronic and relatively irreversible expiratory airflow dysfunction due to some combination of chronic obstructive bronchitis, emphysema, and/or chronic asthma. COPD is caused by a range of environmental and genetic risk factors, including smoking that contributes to the disease.
The prevalence of COPD is increasing worldwide, and COPD has become the fourth leading cause of death in the United States. In the United States, despite the decrease in cigarette smoking in recent decades, both the prevalence of, and the mortality associated with, COPD have increased and are projected to continue to increase for some years yet. Furthermore, COPD is costly, and acute exacerbations, which occur roughly once a year in patients with COPD of moderate or greater severity, constitute the most expensive component.
In COPD, airflow obstruction can occur on the basis of either of two very different pathophysiological processes in the lung: 1) inflammation of the parenchyma resulting in proteolysis of the lung parenchyma and loss of lung elasticity (emphysema); and 2) inflammation, scarring and narrowing of the small airways (“small airway disease”). In an individual patient, one of these processes, which may be controlled by different genetic factors, may predominate although both usually co-exist. Ultimately, both of these processes produce similar patterns of functional impairment: decreased expiratory flow, hyperinflation and abnormalities of gas exchange.
At an early stage of COPD, the following symptoms are found in the lungs of COPD patients: 1) breach of airway epithelium by damaging aerosols, 2) accumulation of inflammatory mucous exudates, 3) infiltration of the airway wall by inflammatory immune cells, 4) airway remodeling/thickening of the airway wall and encroachment on lumenal space, and 5) increased resistance to airflow. During this early stage, smooth muscle contraction and hyper-responsiveness also increase resistance, but the increased resistance is relieved by bronchodilators.
At an advanced stage, COPD patients characteristically develop deposition of fibrous connective tissue in the subepithelial and aventitial compartments surrounding the airway wall. Such peribronchiolar fibrosis contributes to fixed airway obstruction by restricting the enlargement of airway caliber that occurs with lung inflation.
3.5.1. Chronic Bronchitis
Chronic bronchitis is defined as the presence of chronic cough and sputum production for at least three months of two consecutive years in the absence of other diseases recognized to cause sputum production. In chronic bronchitis, epidemiologically the bronchial epithelium becomes chronically inflamed with hypertrophy of the mucus glands and an increased number of goblet cells. The cilia are also destroyed and the efficiency of the mucociliary escalator is greatly impaired. Mucus viscosity and mucus production are increased, leading to difficulty in expectorating. Pooling of the mucus leads to increased susceptibility to infection.
Microscopically there is infiltration of the airway walls with inflammatory cells. Inflammation is followed by scarring and remodeling that thickens the walls and also results in narrowing of the airways. As chronic bronchitis progresses, there is squamous metaplasia (an abnormal change in the tissue lining the inside of the airway) and fibrosis (further thickening and scarring of the airway wall). The consequence of these changes is a limitation of airflow. Repeated infections and inflammation over time leads to irreversible structural damage to the walls of the airways and to scarring, with narrowing and distortion of the smaller peripheral airways.
3.5.2. Emphysema
Emphysema is defined in terms of its pathological features, characterized by abnormal dilatation of the terminal air spaces distal to the terminal bronchioles, with destruction of their wall and loss of lung elasticity. Bullae (blisters larger than 1 cm wide) may develop as a result of overdistention if areas of emphysema are larger than 1 cm in diameter. The distribution of the abnormal air spaces allows for the classification of the two main patterns of emphysema: panacinar (panlobular) emphysema, which results in distension, and destruction of the whole of the acinus, particularly the lower half of the lungs. Centriacinar (centrilobular) emphysema involves damage around the respiratory bronchioles affecting the upper lobes and upper parts of the lower lobes of the lung. Certain forms of emphysema are furthermore known to be associated with fibrosis.
The destructive process of emphysema is predominantly associated with cigarette smoking. Cigarette smoke is an irritant and results in low-grade inflammation of the airways and alveoli. It is known that cigarettes contain over 4,000 toxic chemicals, which affect the balance between the antiprotease and proteases within the lungs, causing permanent damage. Inflammatory cells (macrophages and neutrophils) produce a proteolytic enzyme known as elastase, which destroys elastin, an important component of lung tissue.
The alveoli or air sacs of the lung contain elastic tissue, which supports and maintains the potency of the intrapulmonary airways. The destruction of the alveolar walls allows narrowing in the small airways by loosening the guy ropes that help keep the airways open. During normal inspiration, the diaphragm moves downwards while the rib cage moves outwards, and air is drawn into the lungs by the negative pressure that is created. On expiration, as the rib cage and diaphragm relax, the elastic recoil of the lung parenchyma pushes air upwards and outwards. With destruction of the lung parenchyma, which results in floppy lungs and loss of the alveolar guy ropes, the small airways collapse and air trapping occurs, leading to hyperinflation of the lungs. Hyperinflation flattens the diaphragm, which results in less effective contraction and reduced alveolar efficiency, which in turn leads to further air trapping. Over time the described mechanism leads to severe airflow obstruction, resulting in insufficient expiration to allow the lungs to deflate fully prior to the next inspiration.
3.5.3 Chronic Asthma
Asthma is defined as a chronic inflammatory condition of the airways, leading to widespread and variable airways obstruction that is reversible spontaneously or with treatment. In some patients with chronic asthma, the disease progresses, leading to irreversible airway obstruction, particularly if the asthma is untreated, either because it has not been diagnosed or mismanaged, or if it is particularly severe. Children with asthma have a one in ten chance of developing irreversible asthma, while the risk for adult-onset asthmatics is one in four. Studies also have found that in both children and adults that asthma might lead to irreversible deterioration in lung function if their asthma was not treated appropriately, particularly with corticosteroid therapy.
The airway inflammation in asthma over time can lead to remodeling of the airways through increased smooth muscle, disruption of the surface epithelium increased collagen deposition and thickening of the basement membrane.
3.6 Other Types of Fibrosis
Other types of fibrosis include, without limitation, cystic fibrosis of the pancreas and lungs, injection fibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, and nephrogenic systemic fibrosis.
Cystic fibrosis (CF, mucovidosis, mucovisidosis) is an inherited autosomal recessive disorder. It is one of the most common fatal genetic disorders in the United States, affecting about 30,000 individuals, and is most prevalent in the Caucasian population, occurring in one of every 3,300 live births. The gene involved in cystic fibrosis, which was identified in 1989, codes for a protein called the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR normally is expressed by exocrine epithelia throughout the body and regulates the movement of chloride ions, bicarbonate ions and glutathione into and out of cells. In cystic fibrosis patients, mutations in the CFTR gene lead to alterations or total loss of CFTR protein function, resulting in defects in osmolarity, pH and redox properties of exocrine secretions. In the lungs, CF manifests itself by the presence of a thick mucus secretion which clogs the airways. In other exocrine organs, such as the sweat glands, CF may not manifest itself by an obstructive phenotype, but rather by abnormal salt composition of the secretions (hence the clinical sweat osmolarity test to detect CF patients). The predominant cause of illness and death in cystic fibrosis patients is progressive lung disease. The thickness of CF mucus, which blocks the airway passages, is believed to stem from abnormalities in osmolarity of secretions, as well as from the presence of massive amounts of DNA, actin, proteases and prooxidative enzymes originating from a subset of inflammatory cells, called neutrophils. Indeed, CF lung disease is characterized by early, hyperactive neutrophil-mediated inflammatory reactions to both viral and bacterial pathogens. The hyperinflammatory syndrome of CF lungs has several underpinnings, among which an imbalance between pro-inflammatory chemokines, chiefly IL-8, and anti-inflammatory cytokines, chiefly IL-10, has been reported to play a major role. See Chmiel et al., Clin Rev Allergy Immunol. 3(1):5-27 (2002). Studies have reported that levels of TNF-α, IL-6 and IL-1β were higher in the bronchoalveolar lavage fluid of cystic fibrosis patients, than in healthy control bronchoalveolar lavage fluid (Bondfield, T. L., et al. Am. J. Resp. Crit. Care Med. 152(1):2111-2118, 1995).
Injection fibrosis (IF) is a complication of intramuscular injection often occurring in the quadriceps, triceps and gluteal muscles of infants and children in which subjects are unable to fully flex the affected muscle. It typically is painless, but progressive. Studies have reported that the glycoprotein osteopontin (OPN) plays a role in tissue remodeling (Liaw, L., et al. J. Clin. Invest, 101(7):1469-1478, 1998) and that this proinflammatory mediator induces IL-1β up-regulation in human monocytes and an accompanying enhanced production of TNF-α and IL-6 (Naldini, A., et al. J. Immunol. 177:4267-4270, 2006; Weber, G. F., and Cantor, H. Cytokine Growth Factor Reviews. 7(3):241-248, 1996).
Endomyocardial disease (hyperosinophilic syndrome (HS)) is a disease process characterized by a persistently elevated eosinophil count (1500 eosinophils/mm3) in the blood. HS simultaneously affects many organs. Studies have reported that IL-1β, IL-6 and TNF-α are expressed at high levels in viral-induced myocarditis patients (Satoh, M., et al. Virchows Archiv. 427(5):503-509, 1996). Symptoms may include cardiomyopathy, skin lesions, thromboembolic disease, pulmonary disease, neuropathy, hepatosplenomegaly (coincident enlargement of the liver and spleen), and reduced ventricular size. Treatment may include utilizing corticosteroids to reduce eosinophil levels.
Mediastinal fibrosis (MF) is characterized by invasive, calcified fibrosis centered on lymph nodes that blocks major vessels and airways. MF is a late complication of histoplasmosis. Studies in murine models of fibrosis have reported that IL-10 and TNF-α are elevated significantly (Ebrahimi, B, et al. Am. J. Pathol. 158:2117-2125, 2001).
Myelofibrosis (myeloid metaplasia, chronic idiopathic myelofibrosis, primary myelofibrosis) is a disorder of the bone marrow in which the marrow undergoes fibrosis. Myelofibrosis leads to progressive bone marrow failure. The mean survival is five years and causes of death include infection, bleeding, organ failure, portal hypertension, and leukemic transformation. It has been reported that TNF-α and IL-6 levels are elevated in animal models of viral-induced myelofibrosis (Bousse-Kerdiles, M., et al. Ann. Hematol. 78:434-444, 1999).
Retroperitoneal fibrosis (Ormond's disease) is a disease featuring the proliferation of fibrous tissue in the retroperitoneum. The retroperitoneum is the body compartment containing the kidneys, aorta, renal tract, and other structures. It has been reported that IL-1, IL-6 and TNF-α have key roles in the pathogenesis of retroperitoneal fibrosis (Demko, T., et al, J. Am. Soc. Nephrol. 8:684-688, 1997). Symptoms of retroperitoneal fibrosis may include, but are not limited to, lower back pain, renal failure, hypertension, and deep vein thrombosis.
Nephrogenic systemic fibrosis (NSF, nephrogenic fibrosing dermopathy) involves fibrosis of the skin, joints, eyes and internal organs. NSF may be associated with exposure to gadolinium. Patients develop large areas of hardened skin with fibrotic nodules and plaques. Flexion contractures with an accompanying limitation of range of motion also may occur. NSF shows a proliferation of dermal fibroblasts and dendritic cells, thickened collagen bundles, increased elastic fibers, and deposits of mucin. Some reports have suggested that a proinflammatory state provides a predisposing factor for causing nephrogenic systemic fibrosis (Saxena, S., et al. Int. Urol. Nephrol. 40:715-724, 2008), and that the level of TNF-α is elevated in animal models of nephrogenic systemic fibrosis (Steger-Hartmann, T., et al. Exper. Tox. Pathol. 61(6): 537-552, 2009).
4. Risk Factors
4.1. Primary Risk Factors
4.1.1. Cigarette Smoking
While a number of risk factors for fibrotic airway diseases have been identified (some of which may play a role in their causation), tobacco smoke remains the principal and most important cause of COPD. The greater the number of cigarettes smoked, the greater is the risk of developing fibrotic airwary diseases. An overwhelming majority of people who develop fibrotic airway diseases are smokers, and their lung function decreases faster than that of non-smokers.
The most effective intervention is to stop smoking, preferably at an early stage. Smokers who quit will not recover lost lung function, but the rate of decline may revert to that of a non-smoker. Stopping smoking at an early stage improves the prognosis, regardless of how many attempts are needed to quit. Individual susceptibility to developing fibrotic airwary diseases. in relation to cigarette smoking varies. Approximately 15% of smokers will develop clinically significant COPD, while approximately 50% will never develop any symptoms. The decrease in lung function is gradual, and the disease is usually diagnosed late because patients may adapt to symptoms of shortness of breath, or may not notice the symptoms. Studies have shown that depending on the number of cigarettes smoked per day, 24-47% of smokers develop airflow obstruction. Exposure to passive smoking increases susceptibility to the disease.
4.1.2. Alpha-1 Antitrypsin Deficiency
This rare inherited condition results in the complete absence of one of the key antiprotease protection systems in the lung. It is a recessive disorder affecting 1:4000 of the population. Patients with alpha-1 antitrypsin deficiency are at risk of developing emphysema at an early age-between the ages of 20 and 40 years- and often have a strong family history of the disease. Patients with the deficiency and emphysema inherit one abnormal gene from each parent; that is to say, the parents are carriers of the gene. Such parents will have half the normal levels of the antitrypsin in the blood, which may be enough to protect from developing emphysema. Likewise, all the children of an alpha-1 antitrypsin deficient patient will carry one abnormal gene, but will not be affected. The two common forms of alpha-1 antitrypsin deficiency result from point mutations in the gene that codes for alpha-1 antitrypsin.
4.2. Associated Risk Factors
4.2.1. Environmental Pollution
There is strong evidence that fibrotic airwary diseases may be aggravated by air pollution, but the role of pollution in the etiology of fibrotic airwary diseases is small when compared with that of cigarette smoking. Air pollution with heavy particulate matter, carbon, and sulphur dioxide, which are produced by the burning of coal and petroleum fossil fuels, are important causes or cofactors in the development of fibrotic airwary diseases. These originate mainly from vehicle exhaust emissions, and photochemical pollutants such as ozone, in particular, are to be blamed. Indoor air pollution from biomass fuel burned for cooking and heating in poorly ventilated homes may be an important risk factor for fibrotic airwary diseases, such as COPD, in developing countries, in particular for women.
4.2.2. Occupational Factors
Some occupations in which workers are exposed to coal, silica and cation, such as miners, textile workers and cement workers, are associated with an increased risk of fibrotic airwary diseases. Exposure to cadmium, a heavy metal, and welding fumes has been recognized as a cause of emphysema since the 1950s.
Many dusty occupations are more hazardous than exposure to gas or fumes and are associated with the development of chronic bronchitis and various forms of airway obstructive disease. Shipyard welders and caulkers are also known to have an increased risk of developing fibrotic airwary diseases, as well as those working in the construction industries that are exposed to cement dust.
4.2.3. Childhood Respiratory Infections
Chest infections in the first year of life, such as pneumonia and bronchiolitis, may predispose to the development of COPD in later life. This may be as a result or incomplete development of the respiratory system at birth until lung growth ends in early adulthood. If developing lungs are damaged, maximum potential lung function will not be achieved, producing symptoms of COPD at an early age.
4.3. Other Risk Factors
Other risk factors, which may play a role in causation and/or serves as early symptoms of fibrotic airway diseases, such as pulmonary fibroses, include hypersensitivity pneumonitis (most often resulting from inhaling dust contaminated with bacterial, fungal, or animal products), some typical connective tissue diseases (such as rheumatoid arthritis, systemic lupus erythematosus (SLE) and scleroderma), other diseases that involve connective tissue (such as sarcoidosis and Wegener's granulomatosis), infections, certain medications (e.g. amiodarone, bleomycin, busulfan, methotrexate, and nitrofurantoin), and radiation therapy to the chest.
5. Liver Tissue Remodeling
Liver tissue remodeling is of clinical interest because chronic damage to the liver induces necrosis or the parenchyma with consequent increased deposition of extracellular matrix (ECM) that underlies the occurrence of liver cirrhosis, hepatocellular carcinoma and liver fibrosis (Moshage H., J. Pathol. 1997; 181: 257-266). During remodeling, the liver undergoes rearrangement of the original architecture, with consequent changes in the spatial organization and redefinition of the histological borders (Giannelli G. et al., Histol. Histopathol. 2003; 18: 1267-1274). The break-down of tissue boundaries and rearrangement of tissue architecture seem to be regulated by matrix metalloproteinases (MMPs), a large family of zinc-endopeptidases with proteolytic activity toward ECM components (Giannelli G. et al., Histol. Histopathol. 2003; 18: 1267-1274).
One member of the MMP family, MMP2, is found throughout the human body and possesses a wide spectrum of action toward the ECM (Giannelli G. et al., Histol. Histopathol. 2003; 18: 1267-1274). It is secreted as a pro-enzyme and activated at the cellular surface by a membrane-type MMP (MT1-MMP) with the aid of an MMP inhibitor, tissue inhibitor of metalloproteinase-2 (TIMP2) (Strongin A. Y., et al., Biol. Chem. 1993; 268: 14033-14039). The proteolytic activity of MMP2 is balanced by the presence of TIMP2, which not only activates MMP2 by facilitating binding of MMP2 to MT1-MMP but also inhibits MMP-2, creating a feedback system in the regulation of ECM proteolysis (Fridman R. et al., Biochem. J. 1993; 289(Pt 2): 411-416).
Inflammation and mediators of inflammation, also play a role in liver tissue remodeling (Giannelli G. et al., Histol. Histopathol. 2003; 18: 1267-1274). It has been reported that a number of different cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF) are up-regulated in liver parenchyma during chronic hepatitis (Castilla A. et al., N. Engl. J. Med. 1991; 324: 933-940). The altered network of cytokines is thought to play a role not only in mediating inflammation, but also in modulating the expression of MMPs and TIMPs, thereby influencing the local proteolysis of ECM proteins (Knittel T. et al., J. Hepatol. 1999; 30: 48-60).
5.1 Liver Fibrosis
The pathogenesis of hepatic fibrosis involves significant deposition of fibrillar collagen and other extracellular matrix proteins. It is a dynamic process of wound healing in response to a variety of persistent liver injuries caused by factors such as ethanol intake, viral infection, drugs, toxins, cholestasis and metabolic disorders. Liver fibrosis distorts the hepatic architecture, decreases the number of endothelial cell fenestrations and causes portal hypertension (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231). Key events are the activation and transformation of quiescent hepatic stellate cells into myofibroblast-like cells with the subsequent up-regulation of proteins such as α-smooth muscle actin, interstitial collagens, matrix metalloproteinases, tissue inhibitor of metalloproteinases and proteoglycans (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231). Oxidative stress is a major contributing factor to the onset of liver fibrosis, and it is typically associated with a decrease in the antioxidant defense (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231). Currently, there is no effective therapy for advanced liver fibrosis. In its early stages, liver fibrosis is reversible upon cessation of the causative agent; recent developments in our understanding of the process of hepatic fibrinogenesis suggest that a capacity for recovery from advanced fibrosis is possible (Pellicoro A, et al., Fibrogenesis & Tissue Repair 2012, 5(Suppl 1): S26; Benyon R C and Iredale J P, Gut 2000; 46: 443-446 (April)).
5.1.1 Etiology
Most chronic liver diseases are associated with fibrosis and are characterized by parenchymal damage and inflammation. Alcohol abuse, chronic viral hepatitis (HBV and HCV), obesity, autoimmune hepatitis, parasitic diseases (e.g., schistosomiasis), metabolic disorders (e.g., hemochromatosis and Wilson's disease), biliary disease, persistent exposure to toxins and chemicals and drug-induced chronic liver diseases are the most common causes of hepatic fibrosis (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231).
5.1.1.1 Alcohol Consumption
Alcohol consumption is a predominant etiological factor in the pathogenesis of chronic liver diseases worldwide, resulting in fatty liver, alcoholic hepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma (Miller A M, et al., Alcoholism-Clinical and Experimental Research 2011; 35(5): 787-793). Acetaldehyde, the product of alcohol metabolism, increases the secretion of transforming growth factor β1 (TGFβ1) and induces TGFβ type II receptor expression in hepatic stellate cells (HSC), the key collagen-producing cell within the liver (Anania F A, et al., Arch. Biochem. Biophys. 1996; 331(2): 187-193). TGFβ1 is a critical factor in the progression of alcoholic liver disease (ALD) in patients with steatosis and steatohepatitis and plays a significant role as mediator of alcohol-induced liver fibrosis (Weng H L, et al., Hepatology 2009; 50(1): 230-243). Acetaldehyde also selectively induces phosphorylation of Smad3 but not of Smad2 (Greenwel P, et al., Hepatology 2000; 31(1): 109-116). Both ethanol and acetaldehyde induce the COL1A2 promoter and up-regulate collagen I protein expression (Chen A, Biochem. J. 2002; 368(Pt 3): 683-693).
Hepatic alcohol metabolism generates reactive oxygen species (ROS) causing significant cell death (Parola M and Robino G, J. Hepatol. 2001; 35(2): 297-306). Oxidative stress promotes hepatocyte necrosis and/or apoptosis. Generation of ROS within hepatocytes may be a consequence of an altered metabolic state, as it occurs in nonalcoholic fatty liver disease and non-alcoholic steatohepatitis (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231). Alternatively, it could result from ethanol metabolism as in alcoholic steatohepatitis, with ROS being generated mainly by the mitochondrial electron transport chain, cytochrome P450 isoforms such as cytochrome P4502E1 (CYP2E1), damaged mitochondria, xanthine oxidase, NADPH oxidase and generation of lipid peroxidation-end products (Haorah J, et al., Free Radic. Biol. Med. 2008; 45(11): 1542-1550). In addition, it is known that chronic alcohol consumption lowers glutathione levels, thus contributing to liver injury (Cederbaum A I, World J. Gastroenterol. 2010; 16(11): 1366-1376). ROS-derived mediators released by damaged neighboring cells can directly affect hepatic stellate cell (HSC) behavior. ROS up-regulate the expression of critical genes related to fibrogenesis, including pro-collagen type I, monocyte chemoattractant protein 1 (MCP-1) and tissue inhibitor of metalloproteinase-1 (TIMP1), possibly via activation of a number of critical signal transduction pathways and transcription factors, including c-jun N-terminal kinases (JNKs), activator protein 1 (AP-1) and nuclear factor kappa B (NFκB) (Bataller R, et al., Journal of Clinical Investigation 2005; 115(2): 209-218).
5.1.1.2 Chronic Viral Hepatitis
Chronic hepatitis B virus (HBV) and C virus (HCV) are the most common causes of liver disease worldwide, with an estimated 350 and 170 million individuals, respectively, with chronic infection (Custer B, et al., J. Clin. Gastroenterol. 2004; 38(10): S158-S168). In both cases, there is significant chronic liver injury with subsequent progression to advanced liver fibrosis.
5.1.1.3 Other Causes of Liver Fibrosis
Other factors contributing to hepatic fibrosis are obesity and steatosis, which can lead to nonalcoholic fatty liver disease and to chronic steatohepatitis. Nonalcoholic fatty liver disease has also been reported in non-obese individuals in developing countries (Das K, et al., Hepatology, 2010; 51(5): 1593-1602).
Autoimmune hepatitis, which is the result of anomalous presentation of human leukocyte antigen (HLA) class II in hepatocytes, causes cell-mediated immune responses against the host liver, which can lead to liver fibrosis (Lim Y S, et al., J. Hepatology, 2008; 48(1): 133-139). Parasitic infections, like schistosomiasis, also have been shown to trigger advanced liver fibrosis and portal hypertension (Andersson K L and Chung R T, Curr Treat. Options Gastroenterol., 2007; 10(6): 504-512).
Metabolic disorders, such as hemochromatosis and Wilson's disease, are typically accompanied by chronic hepatitis and fibrosis (Lefkowitch J H, Curr. Opin. Gastroenterol., 2006; 22(3): 198-208). In hereditary hemochromatosis, the excessive absorption and accumulation of iron in tissues and organs, including liver, is related to mutations in the HFE (High-iron) gene (Feder J N, et al., J. Hepatol., 2003; 38(6): 704-709). Pathological accumulation of iron exacerbates oxidative stress resulting in increased lipid peroxidation. This leads to destruction of organelle membranes and in turn, cell death via hepatocyte necrosis and/or apoptosis (Sidorska K, et al., J. Biotech., Computational Bio. And Bionanotech., 2011; 92(1): 54-65). Wilson's disease or hepatolenticular degeneration is a genetic disorder leading to copper accumulation in the liver and is due to a mutation in the APTase (ATP7B) that transports copper (Zhang S, et al., Hum. Mol. Genet., 2011 Aug. 15; 20(16): 3176-3187). Wilson's disease patients are characterized by liver fibrosis caused by severe mitochondrial changes, associated with an increased number of peroxisomes, cytoplasmic lipid droplets and the presence of lipolysosomes, characteristic cytoplasmic bodies formed by lipid vacuoles surrounded by electron-dense lysosomes. Nuclei are frequently involved, with disorganization of the nucleoplasm and with glycogen inclusions (Fanni D, et al., Curr. Med. Chem., 2014; DOI: 10.2174/0929867321666140601163244).
Cholestasis due to bile duct obstruction leads to chronic portal fibrosis and eventually cirrhosis. Moreover, chronic exposure to toxins or chemicals such as N-nitrosodimethylamine, carbon tetrachloride (CCl4) or thioacetamide leads to severe hepatic fibrosis in experimental animal models (George J, et al., Gene Therapy, 2007; 14(10): 790-803; Domoenicali M, et al., J. Hepatol., 2009; 51(6): 991-999; Salguero P R, et al., Lab. Invest. 2008; 88(11): 1192-1203). Exposure to these chemicals in humans is rare and generally occurs in industries where these chemicals are routinely used (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231).
5.2. Cell Types Involved in the Pathogenesis of Liver Fibrosis
5.2.1 Hepatic Stellate Cells
Hepatic stellate cells (HSCs) reside in the space of Disse between hepatocytes and sinusoidal endothelial cells (Friedman S L, Gastroenterology, 2008; 134(6): 1655-1669). Quiescent HSCs are characterized by significant expression of desmin and vitamin A storage. Following liver injury, HSCs lose their vitamin A content, increase expression of α-smooth muscle actin (α-SMA), acquire a myofibroblast-like phenotype, become proliferative, motile, pro-fibrogenic, and contractile, and show abundant rough endoplasmic reticulum (Gressner A M, Kidney International, 1996; 49: S39-S45).
Many factors are known to contribute to activation of HSCs. Damage to hepatocytes and Kupffer cell activation are still considered the primary effectors driving HSC activation (Nieto N, et al., J. Biol. Chem. 2002; 277(12): 9853-9864; Nieto N, Hepatology, 2006; 44(6): 1487-1501). Mediators released from damaged hepatocytes, such as lipid peroxidation products, intermediate metabolites of drugs or hepatotoxins, acetaldehyde and 1-hydroxyethyl radical from alcohol metabolism, as well as ROS (e.g., hydrogen peroxide and superoxide radicals) are strong inducers of HSC activation (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231).
Activated Kupffer cells release ROS and cytokines that are crucial for HSC activation as well (Nieto N, Hepatology, 2006; 44(6): 1487-1501). They are a major source of TGFβ and platelet-derived growth factor (PDGF), two potent profibrogenic cytokines that traditionally have been considered key fibrogenic and proliferative stimuli to HSC (Tsukamoto H, Alcoholism-Clinical and Experimental Research, 1992; 23(5): 911-916). Kupffer cell phagocytic activity generates large amounts of ROS that could further activate HSC and induce their fibrogenic potential. Furthermore, it has been shown that the addition of ethanol and arachidonic acid synergized to activate Kupffer cells modulated the fibrogenic response by a mechanism involving TNFα, reduced glutathione, and TGFβ (Cubero F J and Nieto N, Hepatology, 2008; 48(6): 2027-2039).
Likewise, cytochrome P450 2E1-dependent generation of ROS has been shown to be critical for increased collagen I protein synthesis in co-cultures of hepatocytes and HSCs (Nieto N, et al., J. Biol. Chem., 2002; 277(12): 9853-9864).
5.2.2 Portal Fibroblasts
The portal connective tissue in healthy liver is surrounded by quiescent portal fibroblasts, which constitute a second population of liver cells implicated in portal fibrosis (Tuchweber B, et al., Laboratory Investigation, 1996; 74(1): 265-278). Derived from small portal vessels, they express markers distinct from HSC (e.g. elastin) (Li Z, et al., Hepatology, 2007; 46(4): 1246-1256). Proliferation of biliary cells is often accompanied by proliferation of portal fibroblasts, which form onion-like configurations around biliary structures and acquire a myofibroblast phenotype. These cells are thought to be involved in the early deposition of extracellular matrix (ECM) in portal zones (Desmouliere A, et al., Lab Invest, 1997; 76(6): 765-778).
5.2.3 Bone Marrow-derived Mesenchymal Stem Cells
Evidence suggests that bone marrow-derived stem cells are recruited during both progression and regression of liver fibrosis. During regression from CCl4-induced hepatic fibrosis, bone marrow-derived mesenchymal stem cells migrate into the fibrotic liver, where they can express matrix metalloprotease-13 (MMP13) and MMP9 (Cheng Y J, et al., Life Sci., 2009; 85(13-14): 517-525). In addition, granulocyte colony-stimulating factor (G-CSF) and hepatocyte growth factor (HGF) treatment significantly enhances migration of bone marrow-derived cells into the fibrotic liver and accelerates the regression of liver fibrosis (Higashiyama R, et al., Hepatology, 2007; 45(1): 213-222). Over-expression of hepatocyte growth factor (HGF) together with granulocyte colony-stimulating factor (G-CSF), synergistically stimulate MMP9 expression, which is followed by accelerated resolution of fibrotic scars (Bird T G, et al., Cell Tissue Res., 2008; 331(1): 283-300). Although a significant contribution of bone marrow-derived cells has been shown in human liver fibrosis, it is unclear what type of mesenchymal stem cells are involved (Forbes S J, et al., Gastroenterology, 2004; 126(4): 955-963).
5.2.4 Hepatocytes and Biliary Epithelial Cells
Epithelial-to-mesenchymal transition (EMT) is emerging as a possible source of injury-associated mesenchymal cells derived either from resident hepatocytes or from biliary epithelial cells (Dooley S, et al., Gastroenterology, 2008; 135(2): 642-659; Nitta T, et al., Hepatology, 2008; 48(3): 909-919). The main molecules inducing EMT are TGFβ, epidermal growth factor (EGF), insulin-like growth factor-II (IGF-II) and fibroblast growth factor-2 (FGF-2) (Zeisberg M, et al., J. Biol. Chem., 2007; 282(32): 23337-23347). Hepatocytes that express albumin also express fibroblast-specific protein-1 (FSP 1) in response to CCl4 in vivo or to TGFβ1 in vitro (Zeisberg M, et al., J. Biol. Chem., 2007; 282(32): 23337-23347). It has been reported that hepatocytes express COL1A1 in response to TGFβ1 in vitro, and that Smad signaling mediates EMT (Kaimori A, et al., J. Biol. Chem., 2007; 282(30): 22089-22101).
Biliary epithelial cells have been described to be involved in EMT in liver fibrogenesis. In primary biliary cirrhosis, it has been shown that cells of the bile duct express fibroblast-specific protein-1 (FSP 1) and vimentin, early markers of fibroblasts (Robertson H, et al., Hepatology, 2007; 45(4): 977-981). A consequence of EMT in biliary epithelial cells is the amplification of the pool of portal fibroblasts, contributing significantly to portal fibrosis. In vitro studies with human biliary epithelial cells have confirmed these clinical observations (Rygiel K A, Lab. Invest., 2008; 88(2): 112-123). Thus, EMT could be considered a mechanism participating in the pathogenesis of chronic cholestatic liver disease.
5.2.5 Fibrocytes
Fibrocytes are a circulating, bone marrow-derived, CD34+ cell subpopulation with fibroblast-like properties initially associated with tissue repair in subcutaneous wounds (Bucala R. et al., Mol. Med. 1994; 1(1): 71-81). They comprise a fraction of about 1% of the circulating pool of leukocytes expressing markers of mesenchymal cells (Moore B B, et al., Am. J. Pathol., 2005; 166(3): 675-684). It has been hypothesized that bone marrow-derived circulating CD34+ fibrocytes represent key mediators of liver fibrogenesis in the Abcb4−/− mice, which represents a reproducible, well-characterized non-surgical mouse model for cholangiopathy in humans (Roderfeld M, et al., Hepatology, 2010; 51(1): 267-276).
5.3 Molecular Pathogenesis of Liver Fibrosis
5.3.1 Cell-Cell and Cell-Matrix Interactions
Alterations in normal cell-cell and cell-matrix interactions play a significant role in pathogenesis of liver fibrosis. When normal cell-cell and cell-matrix interactions are altered due to hepatocyte necrosis or invasion of inflammatory cells, new interactions are established that trigger a fibrogenic response (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231). In the fibrotic liver, significant quantitative and qualitative changes occur in the composition of ECM in the periportal and perisinusoidal areas (Zeisberg M, et al., Mol. Cell Biochem., 2006; 283(1-2): 181-189). Fibrotic scars are typically composed of fibrillar collagen type I and III, proteoglycans, fibronectin and hyaluronic acid (George J, et al., International J. Biochem. & Cell Biol., 2004; 36(2): 307-319). As a result, alteration in the physiological architecture of the liver occurs, particularly in the space of Disse, where the low electron-dense ECM is replaced by one rich in fibrillar collagens and fibronectin. This leads to loss of endothelial cell fenestrations, impaired exchange of solutes among neighboring cells, altered hepatocyte function and subsequent non-parenchymal cell damage (Hernandez-Gea V. and Friedman S L, Annu. Rev. Pathol. 2011; 6: 425-456).
5.3.2 Oxidative Stress
The term “oxidative stress” as used herein refers to a disturbance in the balance between the production of reactive oxygen species (free radicals) and antioxidant defenses.
Chronic HBV infection and long-term consumption of alcohol induce cell damage through increased generation of reactive oxygen species (ROS) (Muriel P, Hepatol. Int. 2009; 3(4): 526-536).
Oxidative stress, which favors mitochondrial permeability transition, is able to promote hepatocyte necrosis and/or apoptosis (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231). It has been hypothesized that in some clinically relevant conditions, generation of ROS within hepatocytes may represent an altered metabolic state as in non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, or significant ethanol metabolism as it occurs in alcoholic steatohepatitis (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231).
ROS are generated mainly via the mitochondrial electron transport chain or via activation of cytochrome P450 (mostly cytochrome P450 2E1), NADPH oxidase, xanthine oxidase or via mitochondrial damage. The ROS generated can directly affect HSC and myofibroblast behavior (Tilg H and Hotamisligil G S, Gastroenterology, 2006; 131(3): 934-945; Nieto N, Hepatology, 2006; 44(6): 1487-1501). ROS up-regulate the expression of critical fibrosis-associated genes such as COL1A1, COL1A2, MCP1 and TIMP1 via activation of signal transduction pathways and transcription factors, including JNK, activator protein-1 (AP-1) and NFκB (Bataller R and Brenner D A, Journal of Clinical Investigation, 2005; 115(2): 209-218). ROS generation in HSCs and myofibroblasts occurs in response to several known pro-fibrogenic mediators, including angiotensin II, platelet-derived growth factor (PDGF), TGFβ and leptin (De Minicis S, et al., Gastroenterology, 2006; 131(1): 272-275). Overall, a decrease in the antioxidant defense system, such as GSH, catalase or SOD, in conjunction with enhanced lipid peroxidation, leads to a pro-fibrogenic response by enhancing collagen I protein expression (George J, Clin. Chim. Acta, 2003; 335(1-2): 39-47).
p38 mitogen-activated protein kinases (MAPK) may be essential in the up-regulation of proinflammatory cytokines and can be activated by transforming growth factor β (TGFβ), tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and oxidative stress (Tormos A M, et al., Hepatology, 2013; 57(5): 1950-1961). Substrates of p38 include protein kinases, such as MAPKAP kinase 2 (MK2) and MK5, and transcription factors, such as ATF2, p53, and Mitf (Hui L, et al., Cell Cycle, 2007; 6(20): 2429-2433). These diverse targets mediate the activated p38 signal to various types of cellular functions such as differentiation, apoptosis, cytokine production, and cell cycle control (Nakagawa H and Maeda S, Pathology Research International, 2012; doi: 10.1155/2012/172894). Recent in vivo studies have shown that stress-activated mitogen-activated protein kinase (MAPK) signaling converging on c-Jun NH2-terminal kinase (JNK) and p38 plays a central role in inflammation-mediated liver injury and compensatory hepatocyte proliferation (Sakurai T, et al., PNAS, 2006; 103(28): 10544-10551; Hui L, et al., Nature Genetics, 2007; 39(6): 741-749; Hui L, et al., JCI, 2008; 118(12): 3943-3953; Sakurai T, et al., Cancer Cell, 2008; 14(2): 156-165; Maeda S, Gastroenterology Research and Practice, 2010; doi: 10.1155/2010/367694; Wagner E F and Nebreda A R, Nature Reviews Cancer, 2009; 9(8): 537-549; Min L, et al., Seminars in Cancer Biology, 2011; 21(1): 10-20).
5.3.3 MMPs and TIMPs
The ECM is a highly dynamic milieu subject to constant remodeling. Life-threatening pathological conditions arise when ECM remodeling becomes excessive or uncontrolled. Among the cells involved in hepatic ECM degradation are HSCs, neutrophils and macrophages. MMPs are the main enzymes responsible for ECM degradation (Goto T, et al., Pathophysiology, 2004; 11(3): 153-158). Tissue inhibitors of matrix metalloproteinase-1 (TIMPs) have the ability to inhibit MMPs (Goto T, et al., Pathophysiology, 2004; 11(3): 153-158). Therefore, regulation of the MMP-TIMP balance is crucial for efficient ECM remodeling. The MMP-TIMP balance becomes tipped in response to multiple pro-fibrogenic insults. Activated HSCs not only synthesize and secrete ECM proteins such as collagens type I and type III, but also produce MMP1 and MMP13 (Iredale J P, et al., Clin. Sci. (Lond), 1995; 89(1): 75-81; Knittel T, et al., Hisotchem. Cell Biol., 2000; 113(6): 443-453). However, MMP1 and MMP13 expression decreases as HSC activation progresses, while the activity of other MMPs remains relatively constant, except for MMP2 and MMP9 (Benyon R C and Arthur M J, Semin. Liver Dis. 2001; 21(3): 373-384). The increase in MMP2 activity is associated with distortion of the normal lobular architecture, which further activates HSC (Benyon R C and Arthur M J, Semin. Liver Dis. 2001; 21(3): 373-384). Activated HSCs up-regulate the expression and synthesis of TIMP1 and TIMP2 (Iredale J P, et al., Hepatology, 1996; 24(1): 176-184). TIMP1 not only prevents the degradation of the rapidly increasing ECM by blocking MMPs, but also inhibits the apoptosis of activated HSCs (Murphy F R, et al., J. Biol. Chem., 2002; 277(13): 11069-11076). The net result is the deposition of mature collagen fibers within the space of Disse which results in scarring.
6. Renal Fibrosis
Renal fibrosis is the result of excessive accumulation of extracellular matrix that occurs in nearly every type of chronic kidney disease (Liu Y, Kidney International 2006; 69: 213-217). The progression from chronic kidney disease to renal fibrosis often results in widespread tissue scarring, complete destruction of kidney parenchyma, and end-stage renal failure, a condition that requires dialysis or kidney replacement (Schieppati A et al., Kidney International 2005; 68(Suppl 1): S7-S10). Renal fibrosis represents a failed wound-healing process of kidney tissue after chronic, sustained injury. Several cellular pathways, including mesangial and fibroblast activation, as well as tubular epithelial-mesenchymal transition, have been identified as generating the matrix-producing cells in renal disease conditions (Liu Y, Kidney International 2006; 69: 213-217).
6.1 Cellular Events in Renal Fibrosis
Renal fibrosis is often pathologically described as glomerulosclerosis, tubule-interstitial fibrosis, inflammatory infiltration and loss of renal parenchyma characterized by tubular atrophy, capillary loss and podocyte depletion (Liu Y, Kidney International 2006; 69: 213-217). The underlying cellular events leading to these histologic presentations include mesangial and fibroblast activation, tubular epithelial to mesenchymal transition (EMT), monocyte/macrophage, and T-cell infiltration, and cell apoptosis (Eddy A A, Pediatr. Nephrol. 2000; 15: 290-301; Iwano M and Neilson E G, Curr Opin. Nephrol. Hypertens. 2004; 13: 279-284; Hirschberg R, J. Am. Soc. Nephrol. 2005; 16: 9-11; Liu Y, J. Am. Soc. Nephrol. 2004; 15: 1-12).
6.2 Molecular Pathogenesis of Renal Fibrosis
Although more than a dozen different fibrogenic factors have been implicated in the pathogenesis of renal fibrosis, including various cytokines and hormonal, metabolic, and hemodynamic factors, it is widely accepted that transforming growth factor-β (TGF-β) and its downstream Smad signaling mediators play an essential role (Bottinger E P and Bitzer M, J. Am. Soc. Nephrol. 2002; 13: 2600-2610; Schnaper H W et al., Am. J. Physiol. Renal. Physiol. 2003; 284: F243-F252). In vitro, TGF-β as a sole factor can stimulate mesangial cells, interstitial fibroblasts, and tubular epithelial cells to undergo myofibroblastic activation or transition, to become matrix-producing fibrogenic cells (Liu Y, Kidney International 2006; 69: 213-217; Bottinger E P and Bitzer M, J. Am. Soc. Nephrol. 2002; 13: 2600-2610; Schnaper H W et al., Am. J. Physiol. Renal. Physiol. 2003; 284: F243-F252). Expression of exogenous TGF-β, either via gene delivery in vivo or in transgenic mice, causes renal fibrosis (Liu Y, Kidney International 2006). Conversely, inhibition of TGF-β by multiple strategies suppresses renal fibrotic lesions and prevents progressive loss of kidney function (Liu Y, Kidney International 2006; 69: 213-217; Bottinger E P and Bitzer M, J. Am. Soc. Nephrol. 2002; 13: 2600-2610; Schnaper H W et al., Am. J. Physiol. Renal. Physiol. 2003; 284: F243-F252). TGF-β induction also appears to be a convergent pathway that integrates, directly or indirectly, the effects of many other fibrogenic factors. Some of these, such as angiotensin II and high glucose, act as an upstream TGF-β inducer, whereas others, such as connective tissue growth factor, work as its downstream effector (Liu Y, Kidney International 2006; 69: 213-217; Bottinger E P and Bitzer M, J. Am. Soc. Nephrol. 2002; 13: 2600-2610; Schnaper H W et al., Am. J. Physiol. Renal. Physiol. 2003; 284: F243-F252).
The TGF-β signal is transduced through its cell membrane type I and type II serine/threonine kinase receptors (Bottinger E P and Bitzer M, J. Am. Soc. Nephrol. 2002; 13: 2600-2610). Receptor activation triggers the phosphorylation and activation of its downstream signaling mediators, Smad2 and Smad3 (Liu Y, Kidney International 2006). Phosphorylated Smad2/3 bind to common partner Smad4, and subsequently translocate into nuclei, where they control the transcription of TGF-β-responsive genes (Liu Y, Kidney International 2006; 69: 213-217; Bottinger E P and Bitzer M, J. Am. Soc. Nephrol. 2002; 13: 2600-2610; Schnaper H W et al., Am. J. Physiol. Renal. Physiol. 2003; 284: F243-F252). TGF-β/Smad signaling is regulated at both prereceptor and postreceptor stages through multiple levels of modulation, which include TGF-β gene expression, latent TGF-β activation, its receptor expression, and postreceptor Smad signaling (Liu Y, Kidney International 2006). In the fibrotic kidney, a multitude of mechanisms lead to a hyperactive TGF-β/Smad signaling, including induction of TGF-β expression, enhanced post-translational activation of TGF-β protein and its release from latent complexes. The receptors for TGF-β are also induced in diseased kidney (Liu Y, Kidney International 2006; 69: 213-217).
Smad signaling in normal kidney is tightly constrained by a family of proteins known as Smad transcriptional corepressors, which include SnoN, Ski, and TGIF (Liu Y, Kidney International 2006; 69: 213-217). Through various mechanisms, these Smad antagonists effectively confine Smad-mediated gene transcription, thereby safeguarding the tissue from unwanted TGF-β response (Liu Y, Kidney International 2006; 69: 213-217). Recently, it has been demonstrated that SnoN and Ski are progressively diminished in the fibrotic kidney, suggesting that the loss of Smad antagonists is an important mechanism that amplifies the TGF-β signal (Yang J et al., J. Am. Soc. Nephrol. 2003; 14: 3167-3177).
6.3 Matrix-Degrading Enzymes in Renal Fibrosis
It is generally believed that the excessive matrix accumulation seen in the fibrotic kidney results from both overproduction of matrix components and defects in its degradation. This notion is supported by many observations that plasminogen activator inhibitor-1 and tissue inhibitor of matrix metalloproteinase-1 are often upregulated in the diseased kidney. Renal tissue produces a number of proteases, in which the plasminogen/plasmin and matrix metalloproteinase (MMP) systems constitute a proteolytic network that is capable of degrading all components of matrix proteins (Liu Y, Kidney International 2006).
Given their proteolytic ability, matrix-degrading enzymes are historically considered to reduce matrix accumulation, thereby attenuating renal fibrosis after injury. However, recent genetic studies using knockout mice have painted a different and complex picture of the function of these proteins in relation to fibrotic lesions in vivo (Liu Y, Kidney International 2006). It has been reported that ablation of tissue-type plasminogen activator (tPA) protects the kidney from developing interstitial fibrosis in obstructive nephropathy, which seems to have little to do with its proteolytic activity (Yang J et al., J. Clin. Invest. 2002; 110: 1525-1538). The pathogenic effect of tPA in obstructive nephropathy primarily depends on its ability to induce MMP-9 gene expression (Liu Y, Kidney International 2006). Increased MMP-9 disrupts the integrity of tubular basement membrane, which leads to the promotion of tubular EMT. Further investigations reveal that tPA is able to bind to the cell membrane receptor low-density lipoprotein receptor-related protein-1, induces its phosphorylation on tyrosine residues, triggers intracellular signal transduction, and trans-activates MMP-9 gene expression in renal interstitial fibroblasts (Liu Y, Kidney International 2006).
Plasmin, a serine protease that can directly degrade matrix proteins and activate MMPs, is also thought to be beneficial in reducing renal fibrosis (Liu Y, Kidney International 2006). However, knockout of the plasminogen gene does not aggravate the fibrotic lesions after ureteral obstruction. Instead, mice lacking plasmin display a reduced collagen accumulation, suggesting a significant pathogenic effect of this enzyme (Edgtton K L et al., Kidney International 2004; 66: 68-76).
MMP-2 also is found to be necessary and sufficient to induce tubular EMT in vitro (Cheng S and Lovett D H, Am. J. Pathol. 2003; 162: 1937-1949). Transgenic mice with overexpression of MMP-2 display fibrotic lesions (Liu Y, Kidney International 2006). A recent study also demonstrates that MMP-3 induces Rac1 expression, which causes an increase in cellular reactive oxygen species and promotes EMT (Radisky D C et al., Nature 2005; 436: 123-127).
7. Drug-Induced Kidney Injury
Various in vitro and in vivo studies have shown that the administration of certain drugs acts as a stimulus to trigger various MAPK cascades, which in turn, mediate cellular responses to kidney injury (Naughton C. A., American Family Physician, vol. 78, no. 6, pp. 743-750, 2008). Activation and dysregulation of normal MAPK signaling pathways has been implicated in both acute and chronic kidney injury (Cassidy H et al., Journal of Signal Transduction, vol. 2012, Article ID 463617, 15 pages). Renal biopsies in humans have shown upregulation of MAPKs in a variety of renal conditions, suggesting involvement in human renal disease (Cassidy H et al., Journal of Signal Transduction, vol. 2012, Article ID 463617, 15 pages).
7.1 Antibiotic-induced Kidney Injury
Acute kidney injury (AKI) is a common side effect of antibiotic therapy. Several studies have implicated the MAPK signaling cascade in antibiotic-induced renal injury initiated by several distinct classes of antibiotics.
For example, Volpini et al. showed that MAPKs may be involved in the pathogenesis of acute renal failure following treatment with gentamicin (Volpini R. A. et al., Brazilian Journal of Medical and Biological Research, vol. 39, no. 6, pp. 817-823, 2006). Briefly, the expression of p-p38 MAPK and NF-κB in the kidney during the evolution of tubulointerstitial nephritis and its relationship with histological features and renal function was investigated in gentamicin-treated rats in the presence or absence of an NF-κB inhibitor. Data obtained in this study showed that p38 MAPK expression is increased during the development of gentamicin-induced interstitial nephritis and that such alteration is associated with enhancement of NF-κB expression and the inflammatory process in the renal cortex, suggesting the involvement of the p38 MAPK pathway in gentamicin-induced renal lesions (Volpini R. A. et al., Brazilian Journal of Medical and Biological Research, vol. 39, no. 6, pp. 817-823, 2006). Likewise, Ozbek et al. found that p38-MAPK is upregulated in rat kidneys following gentamicin treatment, and it has been shown that combination treatment with the lipid-lowering drug, atorvastatin, ameliorated gentamicin-induced nephrotoxicity, through inhibition of p38-MAPK and NF-κB expression (Ozbek E. et al., Renal Failure, vol. 31, no. 5, pp. 382-392, 2009).
A study investigating the effects of vancomycin exposure in renal LLCPK1 cells on cell proliferation showed a dose- and time-dependent increase in cell number and total cellular protein (Cassidy H et al., Journal of Signal Transduction, vol. 2012, Article ID 463617, 15 pages; King D. W. and Smith M. A., Toxicology in Vitro, vol. 18, no. 6, pp. 797-803, 2004). These effects were inhibited by pretreatment with the MAPK inhibitor, PD098059, thus preventing vancomycin-induced entry into the cell cycle. This data suggests an association between the cell proliferative effects of vancomycin and the induction of MAPK signaling cascades (Cassidy H et al., Journal of Signal Transduction, vol. 2012, Article ID 463617, 15 pages; King D. W. and Smith M. A., Toxicology in Vitro, vol. 18, no. 6, pp. 797-803, 2004).
7.2 Calcineurin-Inhibitor-Induced Kidney Injury
The calcineurin inhibitors (CNIs) CsA and FK506 are widely used in transplant organ recipients. However, it has been shown that in kidney allografts, CsA and FK506 cause tubulointerstitial, as well as mesangial, fibrosis (Sutherland B. W. et al., Oncogene, vol. 24, no. 26, pp. 4281-4292, 2005). The fibrogenic effect of CNIs in the renal allograft is predominantly mediated by elevated intrarenal expression of TGF-β and subsequent excessive extracellular matrix (ECM) generation (Shihab F. S. et al., American Journal of Kidney Diseases, vol. 30, no. 1, pp. 71-81, 1997; Ignotz R. A. and Massague J., Journal of Biological Chemistry, vol. 261, no. 9, pp. 4337-4345, 1986; Schnaper H. W. et al., American Journal of Physiology, vol. 284, no. 2, pp. F243-F252, 2003). Studies using rat kidney mesangial cells have shown that CsA and FK-506 induce an extremely rapid and dose-dependent increase of Y-Box-binding protein-1 (YB-1) content in a cell type-specific manner. YB-1, a member of the family of cold-shock proteins with mitogenic properties which controls, among other things, TGF-β1 translation in proximal tubular cells, is a downstream target of MAPK ERK1/2 (Lu Z. H. et al., Molecular and Cellular Biology, vol. 25, no. 11, pp. 4625-4637, 2005; Fraser D. J. et al., Kidney International, vol. 73, no. 6, pp. 724-732, 2008; Hanssen L. et al., Journal of Immunology, vol. 187, no. 1, pp. 298-308, 2011).
7.3 Chemotherapeutic Agent-induced Kidney Injury
Cancer chemotherapeutics include, without limitation, alkylating agents (e.g., cyclophosphamide); antimetabolites (e.g., methotrexate); plant alkaloids (e.g., etoposide); anthracyclines (e.g., doxirubicin); antitumor antibiotics (e.g., mitomycin C); platinum compounds (e.g., cisplatin); and taxanes (e.g., taxol) (Chu E. and Devita Jr. V. T., Eds., Physicians' Cancer Chemotherapy Drug Manual 2001, Jones and Bartlett Publishers, London, U K, 2001). Chemotherapeutic agents can cause nephrotoxicity in various ways, with some drugs exerting immediate effects on renal function while others are known to have cumulative effects, causing renal injury after long periods of use (Vogelzang N. J., Oncology, vol. 5, no. 10, pp. 97-105, 1991). For example, cisplatin, one of the most successful antineoplastic agents to date, causes acute kidney injury with clinically measureable nephrotoxicity usually detected 10 days after administration (Cassidy H et al., Journal of Signal Transduction, vol. 2012, Article ID 463617, 15 pages).
It is believed that MAPK plays a pivotal role in cisplatin-induced nephrotoxicity. Arany et al. showed that ERK, and not p38 or JNK/SAPK inhibition, prevented cisplatin induced toxicity (Arany I. et al. American Journal of Physiology, vol. 287, no. 3, pp. F543-F549, 2004). Other studies have shown that pharmacological inhibition of p38 both in vitro and in vivo prevented toxicity (Ramesh G. and Reeves W. B., American Journal of Physiology, vol. 289, no. 1, pp. F166-F174, 2005; Francescato H. D. C. et al., Life Sciences, vol. 84, no. 17-18, pp. 590-597, 2009). It also has been shown that JNK/SAPK inhibition results in a significant reduction in cisplatin-induced nephrotoxicity in vivo (Francescato H. D. C. et al., Nephrology Dialysis Transplantation, vol. 22, no. 8, pp. 2138-2148, 2007). Pabla et al. identified PKCδ as a critical regulator of cisplatin nephrotoxicity. The data showed that during cisplatin nephrotoxicity, Src interacted with, phosphorylated, and activated PKCδ in mouse kidney lysates. After activation, PKCδ regulated MAPKs, but not p53, to induce renal cell apoptosis. Thus, inhibition of PKCδ, pharmacologically or genetically, attenuated kidney cell apoptosis and tissue damage, preserving renal function during cisplatin treatment. Conversely, inhibition of PKCδ enhanced cisplatin-induced cell death in multiple cancer cell lines and, remarkably, enhanced the chemotherapeutic effects of cisplatin in several xenograft and syngeneic mouse tumour models while protecting kidneys from nephrotoxicity (Pabla N. et al., Journal of Clinical Investigation, vol. 121, no. 7, pp. 2709-2722, 2011; Cassidy H et al., Journal of Signal Transduction, vol. 2012, Article ID 463617, 15 pages).
7.4 Nonsteroidal Anti-Inflammatory Drug (NSAID)-Induced Kidney Injury
Nonsteroidal anti-inflammatory drugs (NSAIDs) include carboxylic acids (e.g., aspirin); acetic acids (e.g., diclofenac); propionic acids (e.g., ibuprofen and ketoprofen); and Cox-2 inhibitors (e.g., celecoxib). NSAIDs are known to be nephrotoxic with the spectrum of nephrotoxicity including acute tubular necrosis, acute tubulointerstitial nephritis, glomerulonephritis, renal papillary necrosis, chronic renal failure, salt and water retention, hypertension, and hyperkalaemia (Perazella M. A. and Tray K., American Journal of Medicine, vol. 111, no. 1, pp. 64-67, 2001; Ejaz P. et al., Journal of Association of Physicians of India, vol. 52, pp. 632-640, 2004; Schier R. W. and Henrich W. L., Journal of the American Medical Association, vol. 251, no. 10, pp. 1301-1302, 1984).
Hou et al. investigated the molecular basis of the renal injury induced by NSAIDs by evaluating the expression of the stress marker, heme oxygenase-1 (HO-1), in celecoxib-stimulated glomerular mesangial cells (Ho C. C. et al., Annals of the New York Academy of Sciences, vol. 1042, pp. 235-245, 2005). Treatment with celecoxib resulted in a concentration- and time-dependent increase of HO-1 expression (Ho C. C. et al., Annals of the New York Academy of Sciences, vol. 1042, pp. 235-245, 2005). Conversely treatment with N-acetylcysteine, a free radical scavenger, strongly decreased HO-1 expression, suggesting the involvement of reactive oxygen species (ROS) (Ho C. C. et al., Annals of the New York Academy of Sciences, vol. 1042, pp. 235-245, 2005). Treatment with various MAPK inhibitors showed that only a specific JNK inhibitor attenuated celecoxib-induced HO-1 expression (Ho C. C. et al., Annals of the New York Academy of Sciences, vol. 1042, pp. 235-245, 2005). Kinase assays demonstrated increased phosphorylation and activation of c-JNK following NSAID treatment (Ho C. C. et al., Annals of the New York Academy of Sciences, vol. 1042, pp. 235-245, 2005). Treatment with a PI-3K specific inhibitor prevented the enhancement of HO-1 expression, which correlated with inhibition of the phosphorylation of the PDK-1 downstream substrate Akt/protein kinase B (PKB) (Ho C. C. et al., Annals of the New York Academy of Sciences, vol. 1042, pp. 235-245, 2005). The results of this study suggested that celecoxib-induced HO-1 expression in glomerular mesangial cells may be mediated by ROS via the JNK-PI-3K cascade (Ho C. C. et al., Annals of the New York Academy of Sciences, vol. 1042, pp. 235-245, 2005).
8. Vascular Fibrosis
Vascular fibrosis is characterized by reduced lumen diameter and arterial wall thickening, which is attributed to excessive deposition of extracellular matrix (ECM). It involves proliferation of vascular smooth muscle cell (VSMC), accumulation of ECM and inhibition of matrix degradation. It is associated with the renin-angiotensin-aldosterone system (RAAS), oxidative stress, inflammatory factors, growth factors and imbalance of endothelium-derived cytokine secretion. Specifically, Angiotensin II (Ang II) and aldosterone (the circulating effector hormones of RAAS) have been implicated in the pathophysiology of vascular fibrosis. Transforming growth factor-beta (TGF-beta) has been shown to play a critical role in ECM accumulation and vascular remodeling via up-regulation of connective tissue growth factor (CTGF) and fibroblast growth factor among others. An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) results in collagen accumulation and adverse matrix remodeling. Aberrant expression or function of peroxisome proliferator-activated receptor gamma (PPAR gamma) has also been reported to contribute to the progression of pathological fibrosis and vascular remodeling (Lan T-H et al., Cardiovascular Pathology 22 (2013) 401-407).
It has been suggested that risk factors associated with cardiovascular disease, including hypertension, hyperglycemia, dyslipidemia and hyperhomocysteinemia (HHcy), also act as initiation and progression factors of vascular fibrosis (Lan T-H et al., Cardiovascular Pathology 22 (2013) 401-407).
8.1. Pathogenesis of Vascular Fibrosis
8.1.1. Renin-angiotensin-aldosterone system (RAAS)
RAAS has emerged as one of the essential links in the development of vascular remodeling (Sun Y, Congest. Heart Fail. 2002; 8:11-6). Angiotensin II (Ang II) and aldosterone, the circulating effector hormones of RAAS, are recognized as responsible for the pathophysiology of vascular fibrosis.
Ang II, the principal effector hormone of the RAAS, not only mediates immediate physiological effects of vasoconstriction and blood pressure regulation, but also regulates many processes implicated in vascular pathophysiology, including cell growth/apoptosis of vascular cells, migration of VSMCs, inflammatory responses and ECM remodeling (Ruiz-Ortega M et al., Hypertension 2001; 38:1382-7). Ang II acts through two main specific receptors, AT1 and AT2. Binding of Ang II to AT1 receptor activates a series of signaling cascades, including epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), insulin receptor, c-Src family kinases, Ca2+-dependent proline-rich tyrosine kinase 2 (Pyk2), focal adhesion kinase (FAK) and Janus kinases (JAK), which in turn regulate the Ang II pathologic effects in the vasculature (Hunyady L, Catt K J, Mol Endocrinol 2006; 20: 953-70). AT1 also has been implicated in cell growth and hypertrophy by activating PKC and MAPKs, including ERK1/2, p38 MAPK, and c-Jun NH2-terminal kinase (JNK) (Suzuki H et al., Curr Med Chem Cardiovasc Hematol Agents 2005; 3:305-22). Ang II-mediated EGFR activation occurs in a Src-dependent, redox-sensitive manner, and also by calcium-dependent and -independent pathways (Eguchi S et al., J Biol Chem 1998; 273:8890-6; Ushio-Fukai M et al., Arterioscler Thromb Vasc Biol 2001; 21:489-95). Evidence suggests that Ang II stimulates the phosphorylation of PDGF-β receptor (PDGFβ-R) through the binding of tyrosine-phosphorylated Shc to PDGFβ-R (Heeneman S et al., J Biol Chem 2000; 275: 15926-32). Ang II also has been shown to increase serine phosphorylation of the insulin receptor β-subunit, causing insulin receptor substrate (IRS-1) inactivation both by uncoupling it from downstream effectors (PI3K, PDK1, Akt) and by targeting it for degradation in the proteasomal pathway (Folli F et al., J Clin Invest 1997; 100: 2158-69; Natarajan R et al., Hypertension 1999; 33:378-84). Ang II promotes cellular proliferation and ECM synthesis and induction of several mediators, such as TGF-beta, connective tissue growth factor (CTGF), cytokines (e.g., IL-6, TNF-alpha), and monocyte chemoattractant protein type 1 (MCP-1). It is generally accepted that Ang II-induced ECM production is mainly mediated by up-regulation of TGF-β and its downstream mediator CTGF (Border W A, Noble N A, Hypertension 1998; 31:181-8; Wolf G, Kidney Int 2006; 70:1914-9). Ang II activates the binding of nuclear proteins to the binding site of the activator protein-1 (AP-1) of the TGF-β1 promoter through PKC and p38 MAPK-dependent pathways. Recent studies have shown that Ang II also induces CTGF expression via TGF-beta-independent Smad signaling pathways and increases the production of MMPs involved in matrix degradation, which leads to weakening of vessel walls (Rodriguez-Vita J et al., Circulation 2005; 111:2509-17; Yang F et al., Hypertension 2009; 54:877-84). Ang II is known to stimulate aldosterone production, thus promoting fibrosis and collagen formation (Brilla C G et al., J Mol Cell Cardiol 1994; 26:809-20).
Aldosterone promotes fibrosis and collagen formation through several mechanisms, including upregulation of Ang II receptors, stimulation of TGF-β and CTGF synthesis, stimulation of MMPs activity, and participation in VSMC hypertrophy and endothelial dysfunction (Epstein M, Nephrol Dial Transplant 2003; 18:1984-92). It has been reported that aldosterone stimulates the expression of plasminogen activator inhibitor-1 (PAI-1), the major physiological inhibitor of plasminogen activators, which is implicated in ECM accumulation by inhibiting matrix degradation and inducing CTGF expression via both p38 MAPK cascade and mineralocorticoid receptor (Brown N J, et al., Kidney Int 2000; 58:1219-27; Lee Y S et al., J Korean Med Sci 2004; 19: 805-11). Aldosterone has been shown to induce MMP activity in adult rat ventricular myocytes via activation of the mineralocorticoid receptor, PKC, and reactive oxygen species (ROS)-dependent activation of the MEK/ERK pathway (Rude M K et al., Hypertension 2005; 46:555-61).
8.1.2. Transforming Growth Factor-β (TGF-β)
Transforming growth factor (TGF)-β is a ubiquitously expressed cytokine that belongs to a large superfamily, of which TGF-β1 is most frequently up-regulated in ECM remodeling (Massague J et al., Cell 2000; 103:295-309). TGF-β signals through a heteromeric cell-surface complex of two types of transmembrane serine/threonine kinases, type I receptors and type II receptors (Derynck R et al., Nat Genet 2001; 29:117-2). Type I receptor phosphorylation, which is activated by type II receptors, is essential for the activation of downstream target proteins (Derynck R, Feng X H, Biochim Biophys Acta 1997; 1333:F105-50).
TGF-β predominantly transmits signals through transcription factors called Smads (Massague J et al., Genes Dev 2005; 19: 2783-810.). Smad2 and Smad3 are specific mediators of TGF-beta/activin pathways, whereas Smad1, Smad5 and Smad8 are involved in bone morphogenetic protein (BMP) signaling (especially BMP-7) which inhibits VSMC proliferation via I-Smad (Smad6 and Smad7) activation (Singh N N, Ramji D P, Cytokine Growth Factor Rev 2006; 17:487-99). Studies have shown that overexpression of Smad6 may selectively inhibit BMP receptor signaling whereas Smad7 inhibits both BMP and TGF-beta/activin receptor signaling (Nakao A et al., Trends Mol Med 2002; 8: 361-3). Smad7 expression, resulting in the inhibition of TGF-beta-induced fibronectin, collagen and CTGF expression, can also be induced by activation of the EGF receptor, IL-gamma signaling through signal transducer and activator of transcription (STAT), and TNF-alpha induced activation of NF-Kb (Wang M et al., Arterioscler Thromb Vasc Biol 2006; 26:1503-9; Ikedo H et al., Int J Mol Med 2003; 11: 645-50.).
In addition to the Smad pathway, non-Smad pathways also participate in TGF-beta signaling and serve as nodes for crosstalk with other major signaling pathways (Ruiz-Ortega M et al., Cardiovasc Res 2007; 74: 196-206; Bobik A, Arterioscler Thromb Vasc Biol 2006; 26: 1712-20). The JNK/p38, Erk/MAPK, Rho-like GTPase, and PI3/Akt pathways are believed to reinforce, attenuate or modulate downstream cellular responses possibly accounting for the varying effects of TGF-β (Zhang F et al., J Biol Chem 2009; 284:17564-74). TGF-β participates in the pathogenesis of many cardiovascular diseases, including hypertension, restenosis, atherosclerosis, cardiac hypertrophy, and heart failure. TGF-β plays a critical role in ECM accumulation and vascular remodeling via up-regulating the production of several agents, including growth factors (e.g., CTGF, FGF), related genes (e.g., c-myc, cjun, junBJI, p53) and PAI-1 (Perbal B, Lancet 2004; 363: 62-4; Hayashida T et al., FASEB J 2003; 17:1576-8; Samarakoon R et al., J Cell Physiol 2005; 204:236-46; Seay U et al., J Pharmacol Exp Ther 2005; 315:1005-12). Angiotensin II, mechanical stress, endothelin-I, high glucose, extremes of temperature and pH, steroids, and reactive oxygen species have been found to stimulate TGF-β activation as a mediator of vascular fibrosis (Ruiz-Ortega M et al., Curr Hypertens Rep 2003; 5: 73-9; Li J H et al., Kidney Int 2003; 63: 2010-9.). In addition, MMPs (such as MMP-2 and -9) enhance the release of TGF-β, which stimulates TIMP, ultimately resulting in the inhibition of ECM (i.e., ECM accumulation) and vascular remodeling (Derynck R, Zhang Y E, Nature 2003; 425:577-84).
8.1.3. Connective Tissue Growth Factor (CTGF)
CTGF, a potent pro-fibrotic growth factor, has been implicated in fibroblast proliferation, cellular adhesion, angiogenesis and ECM synthesis (Ruperez M et al., Circulation 2003; 108:1499-505). CTGF promotes VSMC proliferation, migration, and production of ECM, which may play a role in the development and progression of atherosclerosis (Leask A et al., Curr Rheumatol Rep 2002; 4: 136-42). CTGF expression is regulated by several agents, including TGF-β, TNF-α, cAMP, high glucose, dexamethasone, factor VIIa, and mechanical stress (Lau L F, Lam S C, Exp Cell Res 1999; 248: 44-57). TGF-β-induced CTGF production is involved with several signal pathways, including Smads, Ras/MEK/Erk, Ap-1/JNK, PKC, and Tyr, and its expression can be decreased by TNF-α, cAMP, PGE2, IL-4 and PPAR through TGF-β down-regulation (Leask A et al., J Biol Chem 2003; 278: 13008-15; Leask A, Abraham D J, Biochem Cell Biol 2003; 81: 355-63). CTGF also appears to increase the expression of MMP-2 (Fan W H, Karnovsky M J, J Biol Chem 2002; 277: 9800-5). It has been reported that the addition of CTGF to primary mesangial cells induced fibronectin production, cell migration, and cytoskeletal rearrangement, which were associated with recruitment of Src and phosphorylation of p42/44 MAPK and protein kinase B (Crean J K et al, J Biol Chem 2002; 277:44187-94).
8.1.4. Matrix metalloproteinases (MMPs)
MMPs, together with cysteine proteinases, aspartic proteinases, and serine proteinases are proteolytic enzymes involved in ECM and basement membranes (BMs) degradation (Lan T-H et al., Cardiovascular Pathology 22 (2013) 401-407). MMPs play important roles in the physiology of fibrosis, as in liver cirrhosis, fibrotic lung disease, otosclerosis, atherosclerosis, and multiple sclerosis. MMPs are thought to mediate the progression of stable atherosclerotic lesions to an unstable phenotype that is prone to rupture through the destruction of ECM proteins (Lan T-H et al., Cardiovascular Pathology 22 (2013) 401-407). MMP-1, MMP-2, MMP-3 and MMP-9 participate in weakening the connective tissue matrix in the intima, which leads to plaque rupture, acute thrombosis, and SMC proliferation and migration (Amalinei C et al., Rom J Morphol Embryol 2010; 51: 215-28). MMP degradation of the EC basement membrane during diapedesis of inflammatory cells could contribute to a decreased endothelial barrier function with increased influx of plasma proteins. All of these interactions have been shown to increase production of MMPs in macrophages, which may also provide stimuli for MMP production in neighboring cells and mechanisms for activation of secreted MMP zymogens resulting in developing atherosclerotic lesions that may facilitate further structural changes and enable their growth (Galis Z S, Khatri J J, Circ Res 2002; 90: 251-62).
The expression of most MMPs is up-regulated during certain physiological and pathological remodeling processes, and mediated by a variety of inflammatory cytokines, hormones, and growth factors, such as IL-1, IL-6, TNF-α, EGF, PDGF, basic fibroblast growth factor (bFGF), and CD40 [111-113]. TGF-β up-regulates the expression of MMP-2 and MMP-9, while it decreases the expression of MMP-1 and MMP-3 (Mauviel A, J Cell Biochem 1993; 53: 288-95).
Fully activated MMPs can be inhibited by tissue inhibitors of metalloproteinases (TIMPs). An imbalance between myocardial MMPs and TIMPs results in collagen accumulation, adverse matrix remodeling and reactive interstitial fibrosis (Nagase H et al., Cardiovasc Res 2006; 69: 562-73).
8.1.5. Peroxisome Proliferator-Activated Receptor Gamma (PPARγ)
It has been reported that aberrant expression or function of PPARγ contributes to the progression of pathological fibrosis and vascular remodeling (Wei J et al., Curr Opin Rheumatol 2010; 22: 671-6). The antagonistic effects of PPARγ on abrogating TGF-β induced stimulation of collagen and fibronectin synthesis and the secretion of fibrotic growth factors including TGF-β and CTGF revealed the anti-fibrotic role of PPARγ in fibrogenesis (Ghosh A K et al., Arthritis Rheum 2004; 50: 1305-18; Burgess H A et al., Am J Physiol Lung Cell Mol Physiol 2005; 288: L1146-53). It was observed that PPARγ abrogates Smad-dependent collagen stimulation by targeting the p300 transcriptional co-activator (Ghosh A K et al., FASEB J 2009; 23: 2968-77). It has been hypothesized that PPARγ-independent pathways might also be involved in the anti-fibrotic effects triggered by PPARγ ligands, including inhibition of fibroblast migration, adipocyte differentiation and myofibroblast transition (Lan T-H et al., Cardiovascular Pathology 22 (2013) 401-407).
9. Current and Emerging Therapeutic Approaches for Treating Fibrotic Diseases or Conditions
Therapeutic agents currently being used to treat fibrotic diseases are disclosed in Datta et al., British Journal of Pharmacology, 163: 141-172, 2011; incorporated by reference herein). Non-limiting examples of such therapeutic agents include, but are not limited to, purified bovine Type V collagens (e.g., IW-001; ImmuneWorks; United Therapeutics), IL-13 receptor antagonists (e.g., QAX576; Novartis), protein tyrosine kinase inhibitors (e.g., imatinib (Gleevec®); Craig Daniels/Novartis), endothelial receptor antagonists (e.g., ACT-064992 (macitentan); Actelion), dual endothelin receptor antagonists (e.g., bosentan (Tracleer®); Actelion), prostacyclin analogs (inhaled iloprost (e.g., Ventavis®); Actelion), anti-CTGF monoclonal antibodies (e.g., FG-3019), endothelin receptor antagonists (A-selective) (e.g., ambrisentan (Letairis®), Gilead), AB0024 (Arresto), lysyl oxidase-like 2 (LOXL2) monoclonal antibodies (e.g., GS-6624 (formerly AB0024); Gilead), c-Jun N-terminal kinase (JNK) inhibitors (e.g., CC-930; Celgene), Pirfenidone (e.g., Esbriet® (InterMune), Pirespa® (Shionogi)), IFN-γ1b (e.g., Actimmune®; InterMune), pan-neutralizing IgG4 human antibodies against all three TGF-β isoforms (e.g., GC1008; Genzyme), TGF-β activation inhibitors (e.g., Stromedix (STX-100)) recombinant human Pentraxin-2 protein (rhPTX-2) (e.g., PRM151; Promedior), bispecific IL4/IL13 antibodies (e.g., SAR156597; Sanofi), humanized monoclonal antibodies targeting integrin αvβ6 (BIBF 1120; Boehringer Ingelheim), N-acetylcysteine (Zambon SpA), Sildenafil (Viagra®), TNF antagonists (e.g., etanercept (Enbrel®); Pfizer), glucocorticoids (e.g., prednisone, budesonide, mometasone furoate, fluticasone propionate, and fluticasone furoate), bronchodilators (e.g., leukotriene modifers (e.g., Montelukast (SINGUAIR®)), anticholingertic bronchodilators (e.g., Ipratropium bromide and Tiotropium), short-acting β2-agonists (e.g., isoetharine mesylate (Bronkometer®), adrenalin, salbutanol/albuterol, and terbutaline), long-acting β2-agonists (e.g., salmeterol, formoterol, indecaterol (Onbrez®), and combination bronchodilators including, but not limited to, SYMBICORT® (containing both budesonide and formoterol), corticosteroids (e.g., prednisone, budesonide, mometasone furoate), methylated xanthine and its derivatives (e.g., caffeine, aminophylline, IBMX, paraxanthine, pentoxifylline, theobromine, and theophylline), neutrophil elastase inhibitors (e.g., ONO-5046, MR-889, L-694,458, CE-1037, GW-311616, and TEI-8362, and transition-state inhibitors, such as ONO-6818, AE-3763, FK-706, ICI-200,880, ZD-0892 and ZD-8321), phosphodiesterase inhibitors (e.g., roflumilast (DAXAS®; Daliresp®), cilomilast (Ariflo®, SB-207499) and sofosbuvir (Sovaldil®).
9.1. Current Therapies for Liver Fibrosis
Despite significant advances in understanding hepatic fibrosis and in defining targets for therapy, a limited number of anti-fibrotic drugs are approved for clinical use in patients with advanced liver disease (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231). Regression of established fibrosis can be accomplished in selected individuals with chronic liver diseases subjected to effective interferon therapies (Shiratori Y, et al., Ann. Intern. Med. 2000; 132(7): 517-524). However, a large cohort of patients does not respond to conventional treatment and thus remain at risk for progression of fibrosis to cirrhosis (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231).
Several compounds with potential antifibrotic activity, including colchicine and malotilate, have been studied in human trials, but were found not effective (Brenner D A and Alcorn J M, Semin. Liver Dis. 1990; 10(1): 75-83; Takase S, et al., Gastroenterol. Jpn., 1988; 23(6): 639-645). Many agents such as malotilate, genistein, curcumin and silymarin have been shown to be effective in vitro and in experimental animal models (Takase S, et al., Gastroenterol Jpn. 1988; 23(6): 639-645; Fu Y M, et al., Mol. Pharm., 2008; 3(2): 399-409; George J, et al., Biomedicine, 2006; 26(3-4): 18-26). Combination therapy that works at different mechanistic levels may be more appropriate to block HSC activation and the pathogenesis of liver fibrosis (Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231).
10. Kinases and Phosphorylation
Kinases are a ubiquitous group of enzymes that catalyze the phosphoryl transfer reaction from a phosphate donor (usually adenosine-5′-triphosphate (ATP)) to a receptor substrate. Although all kinases catalyze essentially the same phosphoryl transfer reaction, they display remarkable diversity in their substrate specificity, structure, and the pathways in which they participate. A recent classification of all available kinase sequences (approximately 60,000 sequences) indicates kinases can be grouped into 25 families of homologous (meaning derived from a common ancestor) proteins. These kinase families are assembled into 12 fold groups based on similarity of structural fold. Further, 22 of the 25 families (approximately 98.8% of all sequences) belong to 10 fold groups for which the structural fold is known. Of the other 3 families, polyphosphate kinase forms a distinct fold group, and the 2 remaining families are both integral membrane kinases and comprise the final fold group. These fold groups not only include some of the most widely spread protein folds, such as Rossmann-like fold (three or more parallel β strands linked by two a helices in the topological order β-α-β-α-β), ferredoxin-like fold (a common α+β protein fold with a signature βαβα secondary structure along its backbone), TIM-barrel fold (meaning a conserved protein fold consisting of eight α-helices and eight parallel β-strands that alternate along the peptide backbone), and antiparallel β-barrel fold (a beta barrel is a large beta-sheet that twists and coils to form a closed structure in which the first strand is hydrogen bonded to the last), but also all major classes (all α, all β, α+β, α/β) of protein structures. Within a fold group, the core of the nucleotide-binding domain of each family has the same architecture, and the topology of the protein core is either identical or related by circular permutation. Homology between the families within a fold group is not implied.
Group I (23,124 sequences) kinases incorporate protein S/T-Y kinase, atypical protein kinase, lipid kinase, and ATP grasp enzymes and further comprise the protein S/T-Y kinase, and atypical protein kinase family (22,074 sequences). These kinases include: choline kinase (EC 2.7.1.32); protein kinase (EC 2.7.137); phosphorylase kinase (EC 2.7.1.38); homoserine kinase (EC 2.7.1.39); I-phosphatidylinositol 4-kinase (EC 2.7.1.67); streptomycin 6-kinase (EC 2.7.1.72); ethanolamine kinase (EC 2.7.1.82); streptomycin 3′-kinase (EC 2.7.1.87); kanamycin kinase (EC 2.7.1.95); 5-methylthioribose kinase (EC 2.7.1.100); viomycin kinase (EC 2.7.1.103); [hydroxymethylglutaryl-CoA reductase (NADPH2)] kinase (EC 2.7.1.109); protein-tyrosine kinase (EC 2.7.1.112); [isocitrate dehydrogenase (NADP+)] kinase (EC 2.7.1.116); [myosin light-chain] kinase (EC 2.7.1.117); hygromycin-B kinase (EC 2.7.1.119); calcium/calmodulin-dependent protein kinase (EC 2.7.1.123); rhodopsin kinase (EC 2.7.1.125); [beta-adrenergic-receptor] kinase (EC 2.7.1.126); [myosin heavy-chain] kinase (EC 2.7.1.129); [Tau protein] kinase (EC 2.7.1.135); macrolide 2′-kinase (EC 2.7.1.136); I-phosphatidylinositol 3-kinase (EC 2.7.1.137); [RNA-polymerase]-subunit kinase (EC 2.7.1.141); phosphatidylinositol-4,5-bisphosphate 3-kinase (EC 2.7.1.153); and phosphatidylinositol-4-phosphate 3-kinase (EC 2.7.1.154). Group I further comprises the lipid kinase family (321 sequences). These kinases include: I-phosphatidylinositol-4-phosphate 5-kinase (EC 2.7.1.68); I D-myo-inositol-triphosphate 3-kinase (EC 2.7.1.127); inositol-tetrakisphosphate 5-kinase (EC 2.7.1.140); I-phosphatidylinositol-5-phosphate 4-kinase (EC 2.7.1.149); I-phosphatidylinositol-3-phosphate 5-kinase (EC 2.7.1.150); inositol-polyphosphate multikinase (EC 2.7.1.151); and inositol-hexakiphosphate kinase (EC 2.7.4.21). Group I further comprises the ATP-grasp kinases (729 sequences) which include inositol-tetrakisphosphate I-kinase (EC 2.7.1.134); pyruvate, phosphate dikinase (EC 2.7.9.1); and pyruvate, water dikinase (EC 2.7.9.2).
Group II (17,071 sequences) kinases incorporate the Rossman-like kinases. Group II comprises the P-loop kinase family (7,732 sequences). These include gluconokinase (EC 2.7.1.12); phosphoribulokinase (EC 2.7.1.19); thymidine kinase (EC 2.7.1.21); ribosylnicotinamide kinase (EC 2.7.1.22); dephospho-CoA kinase (EC 2.7.1.24); adenylylsulfate kinase (EC 2.7.1.25); pantothenate kinase (EC 2.7.1.33); protein kinase (bacterial) (EC 2.7.1.37); uridine kinase (EC 2.7.1.48); shikimate kinase (EC 2.7.1.71); deoxycytidine kinase (EC 2.7.1.74); deoxyadenosine kinase (EC 2.7.1.76); polynucleotide 5′-hydroxyl-kinase (EC 2.7.1.78); 6-phosphofructo-2-kinase (EC 2.7.1.105); deoxyguanosine kinase (EC 2.7.1.113); tetraacyldisaccharide 4′-kinase (EC 2.7.1.130); deoxynucleoside kinase (EC 2.7.1.145); adenosylcobinamide kinase (EC 2.7.1.156); polyphosphate kinase (EC 2.7.4.1); phosphomevalonate kinase (EC 2.7.4.2); adenylate kinase (EC 2.7.4.3); nucleoside-phosphate kinase (EC 2.7.4.4); guanylate kinase (EC 2.7.4.8); thymidylate kinase (EC 2.7.4.9); nucleoside-triphosphate-adenylate kinase (EC 2.7.4.10); (deoxy)nucleoside-phosphate kinase (EC 2.7.4.13); cytidylate kinase (EC 2.7.4.14); and uridylate kinase (EC 2.7.4.22). Group II further comprises the phosphoenolpyruvate carboxykinase family (815 sequences). These enzymes include protein kinase (HPr kinase/phosphatase) (EC 2.7.1.37); phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32); and phosphoenolpyruvate carboxykinase (ATP) (EC 4.1.1.49). Group II further comprises the phosphoglycerate kinase (1,351 sequences) family. These enzymes include phosphoglycerate kinase (EC 2.7.2.3) and phosphoglycerate kinase (GTP) (EC 2.7.2.10). Group II further comprises the aspartokinase family (2,171 sequences). These enzymes include carbamate kinase (EC 2.7.2.2); aspartate kinase (EC 2.7.2.4); acetylglutamate kinase (EC 2.7.2.8 1); glutamate 5-kinase (EC 2.7.2.1) and uridylate kinase (EC 2.7.4.). Group II further comprises the phosphofructokinase-like kinase family (1,998 sequences). These enzymes include 6-phosphofrutokinase (EC 2.7.1.11); NAD (+) kinase (EC 2.7.1.23); I-phosphofructokinase (EC 2.7.1.56); diphosphate-fructose-6-phosphate I-phosphotransferase (EC 2.7.1.90); sphinganine kinase (EC 2.7.1.91); diacylglycerol kinase (EC 2.7.1.107); and ceramide kinase (EC 2.7.1.138). Group II further comprises the ribokinase-like family (2,722 sequences). These enzymes include: glucokinase (EC 2.7.1.2); ketohexokinase (EC 2.7.1.3); fructokinase (EC 2.7.1.4); 6-phosphofructokinase (EC 2.7.1.11); ribokinase (EC 2.7.1.15); adenosine kinase (EC 2.7.1.20); pyridoxal kinase (EC 2.7.1.35); 2-dehydro-3-deoxygluconokinase (EC 2.7.1.45); hydroxymethylpyrimidine kinase (EC 2.7.1.49); hydroxyethylthiazole kinase (EC 2.7.1.50); I-phosphofructokinase (EC 2.7.1.56); inosine kinase (EC 2.7.1.73); 5-dehydro-2-deoxygluconokinase (EC 2.7.1.92); tagatose-6-phosphate kinase (EC 2.7.1.144); ADP-dependent phosphofructokinase (EC 2.7.1.146); ADP-dependent glucokinase (EC 2.7.1.147); and phosphomethylpyrimidine kinase (EC 2.7.4.7). Group II further comprises the thiamin pyrophosphokinase family (175 sequences) which includes thiamin pyrophosphokinase (EC 2.7.6.2). Group II further comprises the glycerate kinase family (107 sequences) which includes glycerate kinase (EC 2.7.1.31).
Group III kinases (10,973 sequences) comprise the ferredoxin-like fold kinases. Group III further comprises the nucleoside-diphosphate kinase family (923 sequences). These enzymes include nucleoside-diphosphate kinase (EC 2.7.4.6). Group III further comprises the HPPK kinase family (609 sequences). These enzymes include 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (EC 2.7.6.3). Group III further comprises the guanido kinase family (324 sequences). These enzymes include guanidoacetate kinase (EC 2.7.3.1); creatine kinase (EC 2.7.3.2); arginine kinase (EC 2.7.3.3); and lombricine kinase (EC 2.7.3.5). Group III further comprises the histidine kinase family (9,117 sequences). These enzymes include protein kinase (histidine kinase) (EC 2.7.1.37); [pyruvate dehydrogenase (lipoamide)] kinase (EC 2.7.1.99); and [3-methyl-2-oxybutanoate dehydrogenase (lipoamide)] kinase (EC 2.7.1.115).
Group IV kinases (2,768 sequences) incorporate ribonuclease H-like kinases. These enzymes include hexokinase (EC 2.7.1.1); glucokinase (EC 2.7.1.2); fructokinase (EC 2.7.1.4); rhamnulokinase (EC 2.7.1.5); mannokinase (EC 2.7.1.7); gluconokinase (EC 2.7.1.12); L-ribulokinase (EC 2.7.1.16); xylulokinase (EC 2.7.1.17); erythritol kinase (EC 2.7.1.27); glycerol kinase (EC 2.7.1.30); pantothenate kinase (EC 2.7.1.33); D-ribulokinase (EC 2.7.1.47); L-fucolokinase (EC 2.7.1.51); L-xylulokinase (EC 2.7.1.53); allose kinase (EC 2.7.1.55); 2-dehydro-3-deoxygalactonokinase (EC 2.7.1.58); N-acetylglucosamine kinase (EC 2.7.1.59); N-acylmannosamine kinase (EC 2.7.1.60); polyphosphate-glucose phosphotransferase (EC 2.7.1.63); beta-glucoside kinase (EC 2.7.1.85); acetate kinase (EC 2.7.2.1); butyrate kinase (EC 2.7.2.7); branched-chain-fatty-acid kinase (EC 2.7.2.14); and propionate kinase (EC 2.7.2.15).
Group V kinases (1,119 sequences) incorporate TIM β-barrel kinases. These enzymes include pyruvate kinase (EC 2.7.1.40).
Group VI kinases (885 sequences) incorporate GHMP kinases. These enzymes include galactokinase (EC 2.7.1.6); mevalonate kinase (EC 2.7.1.36); homoserine kinase (EC 2.7.1.39); L-arabinokinase (EC 2.7.1.46); fucokinase (EC 2.7.1.52); shikimate kinase (EC 2.7.1.71); 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythriol kinase (EC 2.7.1.148); and phosphomevalonate kinase (EC 2.7.4.2).
Group VII kinases (1,843 sequences) incorporate AIR synthetase-like kinases. These enzymes include thiamine-phosphate kinase (EC 2.7.4.16) and selenide, water dikinase (EC 2.7.9.3).
Group VIII kinases (565 sequences) incorporate riboflavin kinases (565 sequences). These enzymes include riboflavin kinase (EC 2.7.1.26).
Group IX kinases (197 sequences) incorporate dihydroxyacetone kinases. These enzymes include glycerone kinase (EC 2.7.1.29).
Group X kinases (148 sequences) incorporate putative glycerate kinases. These enzymes include glycerate kinase (EC 2.7.1.31).
Group XI kinases (446 sequences) incorporate polyphosphate kinases. These enzymes include polyphosphate kinases (EC 2.7.4.1).
Group XII kinases (263 sequences) incorporate integral membrane kinases. Group XII comprises the dolichol kinase family. These enzymes include dolichol kinases (EC 2.7.1.108). Group XII further comprises the undecaprenol kinase family. These enzymes include undecaprenol kinases (EC 2.7.1.66).
Kinases play indispensable roles in numerous cellular metabolic and signaling pathways, and they are among the best-studied enzymes at the structural level, biochemical level, and cellular level. Despite the fact that all kinases use the same phosphate donor (in most cases, ATP) and catalyze apparently the same phosphoryl transfer reaction, they display remarkable diversity in their structural folds and substrate recognition mechanisms. This probably is due largely to the extraordinary diverse nature of the structures and properties of their substrates.
10.1. Mitogen-Activated Protein Kinase-Activated Protein Kinases (MK2 and MK3)
Different groups of MAPK-activated protein kinases (MAP-KAPKs) have been defined downstream of mitogen-activated protein kinases (MAPKs). These enzymes transduce signals to target proteins that are not direct substrates of the MAPKs and, therefore, serve to relay phosphorylation-dependent signaling with MAPK cascades to diverse cellular functions. One of these groups is formed by the three MAPKAPKs: MK2, MK3 (also known as 3pK), and MK5 (also designated PRAK). Mitogen-activated protein kinase-activated protein kinase 2 (also referred to as “MAPKAPK2”, “MAPKAP-K2”, or “MK2”) is a kinase of the serine/threonine (Ser/Thr) protein kinase family. MK2 is highly homologous to MK3 (approximately 75% amino acid identity). The kinase domains of MK2 and MK3 are most similar (approximately 35% to 40% identity) to calcium/calmodulin-dependent protein kinase (CaMK), phosphorylase b kinase, and the C-terminal kinase domain (CTKD) of the ribosomal S6 kinase (RSK) isoforms. The mk2 gene encodes two alternatively spliced transcripts of 370 amino acids (MK2A) and 400 amino acids (MK2B). The mk3 gene encodes one transcript of 382 amino acids. The MK2- and MK3 proteins are highly homologous, yet MK2A possesses a shorter C-terminal region. The C-terminus of MK2B contains a functional bipartite nuclear localization sequence (NLS) (Lys-Lys-Xaa10-Lys-Arg-Arg-Lys-Lys; SEQ ID NO: 23) that is not present in the shorter MK2A isoform, indicating that alternative splicing determines the cellular localization of the MK2 isoforms. MK3 possesses a similar nuclear localization sequence. The nuclear localization sequence found in both MK2B and MK3 encompasses a D domain (Leu-Leu-Lys-Arg-Arg-Lys-Lys; SEQ ID NO: 24) that studies have shown to mediate the specific interaction of MK2B and MK3 with p38α and p38β. MK2B and MK3 also possess a functional nuclear export signal (NES) located N-terminal to the NLS and D domain. The NES in MK2B is sufficient to trigger nuclear export following stimulation, a process which may be inhibited by leptomycin B. The sequence N-terminal to the catalytic domain in MK2 and MK3 is proline rich and contains one (MK3) or two (MK2) putative Src homology 3 (SH3) domain-binding sites, which studies have shown, for MK2, to mediate binding to the SH3 domain of c-Abl in vitro. Recent studies suggest that this domain is involved in MK2-mediated cell migration.
MK2B and MK3 are located predominantly in the nucleus of quiescent cells while MK2A is present in the cytoplasm. Both MK2B and MK3 are rapidly exported to the cytoplasm via a chromosome region maintenance protein (CRM1)-dependent mechanism upon stress stimulation. Nuclear export of MK2B appears to be mediated by kinase activation, as phosphomimetic mutation of Thr334 within the activation loop of the kinase enhances the cytoplasmic localization of MK2B. Without being limited by theory, it is thought that MK2B and MK3 may contain a constitutively active NLS and a phosphorylation-regulated NES.
MK2 and MK3 appear to be expressed ubiquitously, with predominant expression in the heart, in skeletal muscle, and in kidney tissues.
10.1.1. Activation
Various activators of p38α and p38β potently stimulate MK2 and MK3 activity. p38 mediates the in vitro and in vivo phosphorylation of MK2 on four proline-directed sites: Thr25, Thr222, Ser272, and Thr334. Of these sites, only Thr25 is not conserved in MK3. Without being limited by theory, while the function of phosphorylated Thr25 in unknown, its location between the two SH3 domain-binding sites suggests that it may regulate protein-protein interactions. Thr222 in MK2 (Thr201 in MK3) is located in the activation loop of the kinase domain and has been shown to be essential for MK2 and MK3 kinase activity. Thr334 in MK2 (Thr313 in MK3) is located C-terminal to the catalytic domain and is essential for kinase activity. The crystal structure of MK2 has been resolved and, without being limited by theory, suggests that Thr334 phosphorylation may serve as a switch for MK2 nuclear import and export. Phosphorylation of Thr334 also may weaken or interrupt binding of the C terminus of MK2 to the catalytic domain, exposing the NES and promoting nuclear export.
Studies have shown that, while p38 is capable of activating MK2 and MK3 in the nucleus, experimental evidence suggests that activation and nuclear export of MK2 and MK3 are coupled by a phosphorylation-dependent conformational switch that also dictates p38 stabilization and localization, and the cellular location of p38 itself is controlled by MK2 and possibly MK3. Additional studies have shown that nuclear p38 is exported to the cytoplasm in a complex with MK2 following phosphorylation and activation of MK2. The interaction between p38 and MK2 may be important for p38 stabilization since studies indicate that p38 levels are low in MK2-deficient cells and expression of a catalytically inactive MK2 protein restores p38 levels.
10.1.2. Substrates and Functions
Further studies have shown that the small heat shock protein HSPB1 (also known as heat shock protein 27 or Hsp27), lymphocyte-specific protein LSP-1, and vimentin are phosphorylated by MK2. HSPB1 is of particular interest because it forms large oligomers, which may act as molecular chaperones and protect cells from heat shock and oxidative stress. Upon phosphorylation, HSPB1 loses its ability to form large oligomers and is unable to block actin polymerization, suggesting that MK2-mediated phosphorylation of HSPB1 serves a homeostatic function aimed at regulating actin dynamics that otherwise would be destabilized during stress.
MK3 also was shown to phosphorylate HSPB1 in vitro and in vivo, but its role during stressful conditions has not yet been elucidated. MK2 shares many substrates with MK3. Both enzymes have comparable substrate preferences and phosphorylate peptide substrates with similar kinetic constants. The minimum sequence required for efficient phosphorylation by MK2 was found to be Hyd-Xaa-Arg-Xaa-Xaa-pSer/Thr (SEQ ID NO: 25), where Hyd is a bulky hydrophobic residue.
Experimental evidence supports a role for p38 in the regulation of cytokine biosynthesis and cell migration. The targeted deletion of the mk2 gene in mice suggested that although p38 mediates the activation of many similar kinases, MK2 seems to be the key kinase responsible for these p38-dependent biological processes. Loss of MK2 leads (i) to a defect in lipopolysaccharide (LPS)-induced synthesis of cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and gamma interferon (IFN-γ) and (ii) to changes in the migration of mouse embryonic fibroblasts, smooth muscle cells, and neutrophils.
Consistent with a role for MK2 in inflammatory responses, MK2-deficient mice showed increased susceptibility to Listeria monocytogenes infection and reduced inflammation-mediated neuronal death following focal ischemia. Since the levels of p38 protein also are reduced significantly in MK2-deficient cells, it was necessary to distinguish whether these phenotypes were due solely to the loss of MK2. To achieve this, MK2 mutants were expressed in MK2-deficient cells, and the results indicated that the catalytic activity of MK2 was not necessary to restore p38 levels but was required to regulate cytokine biosynthesis.
The knockout or knockdown studies of MK2 provided strong support that activated MK2 enhances stability of IL-6 mRNA through phosphorylation of proteins interacting with the AU-rich 3′ untranslated region of IL-6 mRNA. In particular, it has been shown that MK2 is principally responsible for phosphorylation of hnRNPA0, an mRNA-binding protein that stabilizes IL-6 RNA. In addition, several additional studies investigating diverse inflammatory diseases have found that levels of pro-inflammatory cytokines, such as IL-6, IL-1β, TNF-α and IL-8, are increased in induced sputum from patients with stable chronic obstructive pulmonary disease (COPD) or from the alveolar macrophages of cigarette smokers (Keatings V. et al, Am J Resp Crit Care Med, 1996, 153:530-534; Lim, S. et al., J Respir Crit Care Med, 2000, 162:1355-1360). Elevated levels of pro-inflammatory cytokines, such as interleukin-8 (IL-8) and interleukin-6 (IL-6), as well as related downstream cell adhesion molecules (CAMs) such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinases such as matrix metalloproteinase-7 (MMP-7), and signaling molecules such as 5100 calcium-binding protein A12 (S100A12, also known as calgranulin C), in the peripheral blood have been found to be associated with mortality, lung transplant-free survival, and disease progression in patients with idiopathic pulmonary fibrosis (Richards et al., Am J Respir Crit Care Med, 2012, 185: 67-76; Richards, T. et al., Am J Respir Crit Care Med, 181: A1120, 2010; Moodley, Y. et al., Am J Respir Cell Mol Biol., 29(4): 490-498, 2003). Taken together, these studies implicate that elevated levels of inflammatory cytokines induced by MK2 activation may be involved in the pathogenesis of airway or lung tissue diseases; and suggest a potential for anti-cytokine therapy for treating airway or lung tissue diseases, such as idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease (COPD) (Chung, K., Eur Respir J, 2001, 18: Suppl. 34: 50-59).
10.1.3. Regulation of mRNA Translation
Previous studies using MK2 knockout mice or MK2-deficient cells have shown that MK2 increases the production of inflammatory cytokines, including TNF-α, IL-1, and IL-6, by increasing the rate of translation of its mRNA. No significant reductions in the transcription, processing, and shedding of TNF-α could be detected in MK2-deficient mice. The p38 pathway is known to play an important role in regulating mRNA stability, and MK2 represents a likely target by which p38 mediates this function. Studies utilizing MK2-deficient mice indicated that the catalytic activity of MK2 is necessary for its effects on cytokine production and migration, suggesting that, without being limited by theory, MK2 phosphorylates targets involved in mRNA stability. Consistent with this, MK2 has been shown to bind and/or phosphorylate the heterogeneous nuclear ribonucleoprotein (hnRNP) A0, tristetraprolin, the poly (A)-binding protein PABP1, and HuR, a ubiquitously expressed member of the elav (embryonic-lethal abnormal visual in Drosophila melanogaster) family of RNA-binding protein. These substrates are known to bind or copurify with mRNAs that contain AU-rich elements in the 3′ untranslated region, suggesting that MK2 may regulate the stability of AU-rich mRNAs such as TNF-α. It currently is unknown whether MK3 plays similar functions, but LPS treatment of MK2-deficient fibroblasts completely abolished hnRNP AO phosphorylation, suggesting that MK3 is not able to compensate for the loss of MK2.
MK3 participates with MK2 in phosphorylation of the eukaryotic elongation factor 2 (eEF2) kinase. eEF2 kinase phosphorylates and inactivates eEF2. eEF2 activity is critical for the elongation of mRNA during translation, and phosphorylation of eEF2 on Thr56 results in the termination of mRNA translation. MK2 and MK3 phosphorylation of eEF2 kinase on Ser377 suggests that these enzymes may modulate eEF2 kinase activity and thereby regulate mRNA translation elongation.
10.1.4. Transcriptional Regulation by MK2 and MK3
Nuclear MK2, similar to many MKs, contributes to the phosphorylation of cAMP response element binding (CREB), serum response factor (SRF), and transcription factor ER81. Comparison of wild-type and MK2-deficient cells revealed that MK2 is the major SRF kinase induced by stress, suggesting a role for MK2 in the stress-mediated immediate-early response. Both MK2 and MK3 interact with basic helix-loop-helix transcription factor E47 in vivo and phosphorylate E47 in vitro. MK2-mediated phosphorylation of E47 was found to repress the transcriptional activity of E47 and thereby inhibit E47-dependent gene expression, suggesting that MK2 and MK3 may regulate tissue-specific gene expression and cell differentiation.
10.1.5. Other Targets of MK2 and MK3.
Several other MK2 and MK3 substrates also have been identified, reflective of the diverse functions of MK2 and MK3 in several biological processes. The scaffolding protein 14-3-3ζ is a physiological MK2 substrate. Studies indicate 14-3-3ζ interacts with a number of components of cell signaling pathways, including protein kinases, phosphatases, and transcription factors. Additional studies have shown that MK2-mediated phosphorylation of 14-3-3ζ on Ser58 compromises its binding activity, suggesting that MK2 may affect the regulation of several signaling molecules normally regulated by 14-3-3ζ.
Additional studies have shown that MK2 also interacts with and phosphorylates the p16 subunit of the seven-member Arp2 and Arp3 complex (p16-Arc) on Ser77. p16-Arc has roles in regulating the actin cytoskeleton, suggesting that MK2 may be involved in this process.
MK2 and MK3 also may phosphorylate 5-lipoxygenase. 5-lipoxygenase catalyzes the initial steps in the formation of the inflammatory mediator leukotrienes. Tyrosine hydroxylase, glycogen synthase, and Akt also were shown to be phosphorylated by MK2. Finally, MK2 phosphorylates the tumor suppressor protein tuberin on Ser1210, creating a docking site for 14-3-3ζ. Tuberin and hamartin normally form a functional complex that negatively regulates cell growth by antagonizing mTOR-dependent signaling, suggesting that p38-mediated activation of MK2 may regulate cell growth by increasing 14-3-3ζ binding to tuberin.
10.2. Kinase Inhibition
The eukaryotic protein kinases constitute one of the largest superfamilies of homologous proteins that are related by virtue of their catalytic domains. Most related protein kinases are specific for either serine/threonine or tyrosine phosphorylation. Protein kinases play an integral role in the cellular response to extracellular stimuli. Thus, stimulation of protein kinases is considered to be one of the most common activation mechanisms in signal transduction systems. Many substrates are known to undergo phosphorylation by multiple protein kinases, and a considerable amount of information on primary sequence of the catalytic domains of various protein kinases has been published. These sequences share a large number of residues involved in ATP binding, catalysis, and maintenance of structural integrity. Most protein kinases possess a well conserved 30-32 kDa catalytic domain.
Studies have attempted to identify and utilize regulatory elements of protein kinases. These regulatory elements include inhibitors, antibodies, and blocking peptides.
10.2.1. Inhibitors
Enzyme inhibitors are molecules that bind to enzymes thereby decreasing enzyme activity. The binding of an inhibitor may stop a substrate from entering the active site of the enzyme and/or hinder the enzyme from catalyzing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically (e.g., by modifying key amino acid residues needed for enzymatic activity) so that it no longer is capable of catalyzing its reaction. In contrast, reversible inhibitors bind non-covalently and different types of inhibition are produced depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both.
Enzyme inhibitors often are evaluated by their specificity and potency. The term “specificity” as used in this context refers to the selective attachment of an inhibitor or its lack of binding to other proteins. The term “potency” as used herein refers to an inhibitor's dissociation constant, which indicates the concentration of inhibitor needed to inhibit an enzyme.
Inhibitors of protein kinases have been studied for use as a tool in protein kinase activity regulation. Inhibitors have been studied for use with, for example, cyclin-dependent (Cdk) kinase, MAP kinase, serine/threonine kinase, Src Family protein tyrosine kinase, tyrosine kinase, calmodulin (CaM) kinase, casein kinase, checkpoint kinase (Chkl), glycogen synthase kinase 3 (GSK-3), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase 1 (MEK), myosin light chain kinase (MLCK), protein kinase A, Akt (protein kinase B), protein kinase C, protein kinase G, protein tyrosine kinase, Raf kinase, and Rho kinase.
10.2.2. Blocking Peptides
A peptide is a chemical compound that is composed of a chain of two or more amino acids whereby the carboxyl group of one amino acid in the chain is linked to the amino group of the other via a peptide bond. Peptides have been used inter alia in the study of protein structure and function. Synthetic peptides may be used inter alia as probes to see where protein-peptide interactions occur. Inhibitory peptides may be used inter alia in clinical research to examine the effects of peptides on the inhibition of protein kinases, cancer proteins and other disorders.
The use of several blocking peptides has been studied. For example, extracellular signal-regulated kinase (ERK), a MAPK protein kinase, is essential for cellular proliferation and differentiation. The activation of MAPKs requires a cascade mechanism whereby MAPK is phosphorylated by an upstream MAPKK (MEK) which then, in turn, is phosphorylated by a third kinase MAPKKK (MEKK). The ERK inhibitory peptide functions as a MEK decoy by binding to ERK.
Other blocking peptides include autocamtide-2 related inhibitory peptide (AIP). This synthetic peptide is a highly specific and potent inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII). AIP is a non-phosphorylatable analog of autocamtide-2, a highly selective peptide substrate for CaMKII. AIP inhibits CaMKII with an IC50 of 100 nM (IC50 is the concentration of an inhibitor required to obtain 50% inhibition). The AIP inhibition is non-competitive with respect to syntide-2 (CaMKII peptide substrate) and ATP but competitive with respect to autocamtide-2. The inhibition is unaffected by the presence or absence of Ca2+/calmodulin. CaMKII activity is inhibited completely by AIP (1 μM) while PKA, PKC and CaMKIV are not affected.
Other blocking peptides include cell division protein kinase 5 (Cdk5) inhibitory peptide (CIP). Cdk5 phosphorylates the microtubule protein tau at Alzheimer's Disease-specific phospho-epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to amyloid β peptides. Upon neuronal infections with CIP, CIPs selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. The reasons for the specificity demonstrated by CIP are not fully understood.
Additional blocking peptides have been studied for extracellular-regulated kinase 2 (ERK2), ERK3, p38/HOG1, protein kinase C, casein kinase II, Ca2+/calmodulin kinase IV, casein kinase II, Cdk4, Cdk5, DNA-dependent protein kinase (DNA-PK), serine/threonine-protein kinase PAK3, phosphoinositide (PI)-3 kinase, PI-5 kinase, PSTAIRE (the cdk highly conserved sequence), ribosomal S6 kinase, GSK-4, germinal center kinase (GCK), SAPK (stress-activated protein kinase), SEK1 (stress signaling kinase), and focal adhesion kinase (FAK).
11. Cell Penetrating Peptides (CPPs)
Cell penetrating peptides (CPPs) are a class of peptides capable of penetrating the plasma membrane of mammalian cells and of transporting compounds of many types and molecular weights across the membrane. These compounds include effector molecules, such as proteins, DNA, conjugated peptides, oligonucleotides, and small particles such as liposomes. When CPPs are chemically linked or fused to other proteins, the resulting fusion proteins still are able to enter cells. Although the exact mechanism of transduction is unknown, internalization of these proteins is not believed to be receptor-mediated or transporter-mediated. CPPs are generally 10-16 amino acids in length and may be grouped according to their composition, such as, for example, peptides rich in arginine and/or lysine.
The use of CPPs capable of transporting effector molecules into cells has become increasingly attractive in the design of drugs as they promote the cellular uptake of cargo molecules. These cell-penetrating peptides, generally categorized as amphipathic (meaning having both a polar and a nonpolar end) or cationic (meaning of or relating to containing net positively charged atoms) depending on their sequence, provide a non-invasive delivery technology for macromolecules. CPPs often are referred to as “Trojan peptides,” “membrane translocating sequences,” “protein transduction domains (PTDs),” or “cell permeable proteins (CPPs).” CPPs also may be used to assist novel HSPB1 kinase inhibitors to penetrate cell membranes. (see U.S. application Ser. No. 11/972,459, entitled “Polypeptide Inhibitors of HSPB1 Kinase and Uses Therefor,” filed Jan. 10, 2008, and Ser. No. 12/188,109, entitled “Kinase Inhibitors and Uses Thereof,” filed Aug. 7, 2008, the contents of each application are incorporated by reference in their entirety herein).
11.1. Viral CPP Containing Proteins
The first proteins to be described as having transduction properties were of viral origin. These proteins still are the most commonly accepted models for CPP action. Among the cell-penetrating peptides, the arginine-rich cell-penetrating peptides, including but not limited to TAT peptide, have been the most widely studied (El-Sayed, A. et al., AAPS J. 11, 13-22, 2009; Wender, P. et al., Adv. Drug Deliv. Rev. 60, 452-472, 2008).
TAT (HIV-1 trans-activator gene product) is an 86-amino acid polypeptide, which acts as a powerful transcription factor of the integrated HIV-1 genome. TAT acts on the viral genome stimulating viral replication in latently infected cells. The translocation properties of the TAT protein enable it to activate quiescent infected cells, and it may be involved in priming of uninfected cells for subsequent infection by regulating many cellular genes, including cytokines. The minimal CPP of TAT is the 9 amino acid protein sequence RKKRRQRRR (TAT49-57; SEQ ID NO: 20). Studies utilizing a longer fragment of TAT demonstrated successful transduction of fusion proteins up to 120 kDa. The addition of multiple TAT-CPP as well as synthetic TAT derivatives has been demonstrated to mediate membrane translocation. TAT CPP containing fusion proteins have been used as therapeutic moieties in experiments involving cancer, transporting a death-protein into cells, and disease models of neurodegenerative disorders.
VP22 is the HSV-1 tegument protein, a structural part of the HSV virion. VP22 is capable of receptor independent translocation and accumulates in the nucleus. This property of VP22 classifies the protein as a CPPs containing peptide. Fusion proteins comprising full length VP22 have been translocated efficiently across the plasma membrane.
11.2. Homeoproteins with Intercellular Translocation Properties
Homeoproteins are highly conserved, transactivating transcription factors involved in morphological processes. They bind to DNA through a specific sequence of 60 amino acids. The DNA-binding homeodomain is the most highly conserved sequence of the homeoprotein. Several homeoproteins have been described to exhibit CPP-like activity; they are capable of efficient translocation across cell membranes in an energy-independent and endocytosis-independent manner without cell type specificity.
The Antennapedia protein (Antp) is a trans-activating factor capable of translocation across cell membranes; the minimal sequence capable of translocation is a 16 amino acid peptide corresponding to the third helix of the protein's homeodomain (HD). The internalization of this helix occurs at 4° C., suggesting that this process is not endocytosis dependent. Peptides up to 100 amino acids produced as fusion proteins with AntpHD penetrate cell membranes.
Other homeodomains capable of translocation include Fushi tarazu (Ftz) and Engrailed (En) homeodomain. Many homeodomains share a highly conserved third helix.
11.3. Human CPPs
Human CPPs may circumvent potential immunogenicity issues upon introduction into a human patient. Peptides with CPPs sequences include: Hoxa-5, Hox-A4, Hox-B5, Hox-B6, Hox-B7, HOX-D3, GAX, MOX-2, and FtzCPP. These proteins all share the sequence found in AntpCPPs. Other CPPs include Islet-1, interleukin-1, tumor necrosis factor, and the hydrophobic sequence from Kaposi-fibroblast growth factor or FGF-4) signal peptide, which is capable of energy-, receptor-, and endocytosis-independent translocation. Unconfirmed CPPs include members of the Fibroblast Growth Factor (FGF) family.
12. MK2 Inhibitors and Treatment of Fibrotic Diseases or Conditions
Mitogen-activated protein kinase activated protein kinase 2 (MAPKAPK2 or MK2), a serine/threonine kinase substrate downstream of p38MAPK, has been implicated in many inflammatory diseases that are complicated by scarring and fibrosis (Lopes, L. et al., Biochem Biophys Res Commun., 382(3):535-9, 2009). These include, but are not limited to, cancer, intimal hyperplasia, organ fibrosis, abdominal adhesions, inflammatory bowel disease, and rheumatoid arthritis. In addition to idiopathic pulmonary fibrosis (IPF), other disorders that involve inflammation and fibrosis and impact the lung include acute lung injury (ALI), organ transplant rejection (with lung transplant also a later-stage treatment for IPF), organ failure secondary to sepsis, acute lung failure, auto-immune diseases such as scleroderma, and chronic pulmonary obstructive disease (COPD).
The development of fibrosis is known to require inflammation, proliferation and recruitment of fibroblast that results in cells of myofibroblastic phenotype (Horowitz J. et al., Semin Respir Crit Care Med., 27(6):600-612, 2006). MK2 has been shown to control gene expression at transcriptional and post-transcriptional levels (Neininger A. et al., J Biol Chem. 2002; 277(5):3065-8, Thomas T. et al., J Neurochem., 105(5): 2039-52, 2008; Johansen C. et al., J Immunol., 176(3):1431-8, 2006; Rousseau S. et al., EMBO J. 21(23):6505-14, 2002) as well as cytoskeletal architecture (Lopes, L. et al., Biochem Biophys Res Commun., 382(3):535-9, 2009). In addition, it was shown that activated MK2 increases translation and stability of inflammatory cytokine mRNAs and causes actin reorganization; and that inhibition of MK2 is associated with reduced inflammation (Ward, B. et al., J Surg Res., 169(1):e27-36, 2011) and myofibroblast differentiation (Lopes, L. et al., Biochem Biophys Res Commun., 382(3):535-9, 2009).
Together, these data suggest that inhibition of MK2 may provide therapeutic benefits to patients with fibrotic disorders or conditions, for example, idiopathic pulmonary fibrosis (IPF), acute lung injury (ALI), hepatic fibrosis, renal fibrosis, vascular fibrosis, and transplant rejection. In this respect, the described invention offers an approach to intervene in the process of inflammation and fibrosis using cell-penetrating, peptide base inhibitors of MK2.